Hollow biodegradable nanospheres and nanoshells for delivery of therapeutic and/or imaging molecules

ABSTRACT

A polymeric hollow nanoshell or nanosphere for release of an agent is described, wherein the hollow nanosphere comprises at least one biodegradable polymer, characterised in that the polymer is cross-linked. The biodegradable mono-disperse nanospheres described are suitable for use as carriers of biomolecules, therapeutic agents and/or imaging agents.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.12/886,492, filed Sep. 20, 2010, which is a continuation-in-part ofInternational Application No. PCT/EP2009/053258, filed Mar. 19, 2009,which in turn claims priority to Irish Application No. 2008/0211, filedMar. 20, 2008, the contents of each of which are hereby incorporated byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates to biodegradable mono-dispersednanospheres and nanoshells for use as carriers of biomolecules,therapeutic agents and/or imaging agents or for use in other nanoshelland nanosphere applications. In particular, the invention relates tobiodegradable nanospheres and nanoshells which may be modified to targetdrug or agent delivery to a specific site.

BACKGROUND OF THE INVENTION

There is a significant clinical need for novel methods of detection andtreatment of gene disorders and diseases, such as cancer, that offerimproved sensitivity, specificity, and cost-effectiveness. The object ofany gene or drug therapy is to safely deliver therapeutic agents insidethe cell. It has been more than 12 years since the first gene therapytrial, and to date, after much intense research and more than 600clinical trials, no gene therapy has been approved. The main hurdle toovercome in this field is the lack of efficient, specific and safenucleic acid (DNA, miRNA or sRNA) delivery systems. For successfuldelivery, the therapeutic agent or imaging agent/nucleic acid should bedelivered to the target tissue or cell type. Once delivered to the localtarget site, the agent should be able to enter the cell for imaging orrepair. The delivery device should be smaller than the size of thetarget cell in order to achieve entry into the cell. Chan et al., NanoLett.; 6(4), 662-668 (2006) have reported the effect of the size of adelivery device on cell penetration. According to the research, a devicewhich is nano-meters in size could enter a cell whose size is higherthan micrometers.

In recent years, polymeric micelles have been the object of growingscientific attention. Polymeric micelles have emerged as a potentialcarrier for biomolecules for several reasons. They can solubilisebiomolecules in their inner core, they offer attractive characteristicsin spatial dimensions (>100 nm) for cell entry and they have thecapacity to evade scavenging by the mononuclear phagocyte system.Advantageously, micelle forming polymers usually contain blockco-polymers, which are found in sequences of hydrophobic blocks of thecopolymer comprised of poly(caprolactone), poly(d,l-lactide) orpoly(propylene) with a hydrophilic block of poly(ethylene glycol) PEGsegments. With such polymers, it is possible to design a system thatdoes not precipitate out of solution, is stable and contains a largenumber of distinct microscopic domains. These domains usually possesshydrophobic cores and highly hydrated hydrophilic shells or coronas.Current micellar systems however are only responsive to a limited extentto their biological environment and cannot be functionalized forspecific delivery to a target cell.

Recently, nanoparticles are thought to have potential as novel probesfor both diagnostic (e.g. imaging) and therapeutic purposes (e.g. drugdelivery). In particular, specialized nanoparticle systems known asnanoshells, have shown promise in delivering genes to cells. However,these nanoshells are generally made of non-biodegradable materials, forexample, synthetic polymers such as polyallylamine hydrochloride orinorganic material such as gold or silica, which have long-termbiocompatibility concerns.

A number of other techniques have been investigated to directtherapeutics and diagnostic agents to tumours. These have includedtargeting of tumour cell surface molecules, targeting regions ofactivated endothelium, utilizing the dense and leaky vasculatureassociated with tumours and taking advantage of the enhanced metabolicand proteolytic activities associated with tumours. Antibody labellinghas been used to achieve cell-selective targeting of therapeutic anddiagnostic agents. A number of approaches have been taken forantibody-targeting of therapeutic agents. These have included directconjugation of antibodies to drugs such as interferon alpha, tumournecrosis factor and saporin. Antibody conjugation has also been used fortumour-targeting of radioisotopes for radioimmunotherapy andradioimmunodetection. Currently, there is a commercial product fordetection of prostate cancer (ProstaScint) that is an antibody againstprostate-specific membrane antigen conjugated to a scintigraphic target.

International Patent Publication No. WO 01/64164 describes labellednanocapsules comprising DNA within surfactant micelles and encapsulatedwithin a biocompatible hydrophilic polymer.

Virus particles have been developed that display single chain antibodieson their surface, allowing specific targeting of a wide variety of celltypes. In order to target regions of activated endothelium,immunoliposomes have been made with antibodies to E-selectin on theirsurfaces. Recently tumours have been imaged using protease-activatednear infrared fluorescent probes.

Over the past several years, there has been increasing interest incombining emerging optical technologies with the development of novelexogenous contrast agents, designed to probe the molecular specificsignatures of cancer and to improve the detection limits and theclinical effectiveness of optical imaging.

Sokolov et al (Cancer Research 63, 1999-2004 (2003)) recentlydemonstrated the use of gold colloid conjugated to antibodies to theepidermal growth factor receptor (EGFR) as scattering contrast agentsfor biomolecular optical imaging of cervical cancer cells and tissuespecimens. In addition, multiple groups including Bruchez et al.(Science 281, 2013-2016 (1998)) and Akerman et al. (Proc. Natl. Acad.Sci. U.S.A., 99, 12617-12621 (2002)) have disclosed optical imagingapplications of nanocrystal bioconjugates. More recently, interest hasdeveloped in the creation of nanotechnology-based platform technologies,which couple molecular specific early detection strategies withappropriate therapeutic intervention and monitoring capabilities.

Nedeljkovic and Patel (Appl. Phys. Lett., 58, 2461-63, (1991)) disclosedsilver-coated silver bromide particles that are produced by intense UVirradiation of a mixture of silver bromide, silver, sodiumdodecylsulfate (SDS) and ethylenediaminetetraacetic acid (EDTA). TheNeideljkovic particles range in size from approximately 10 to 40 nm andare irregularly shaped as determined by transmission electronmicroscopy. Predictably, the spectra obtained from these particlepreparations are extremely broad.

U.S. Pat. No. 5,023,139 discloses theoretical calculations indicatingthat metal-coated, semiconducting, nanometer-sized particles shouldexhibit third-order non-linear optical susceptibility relative touncoated dielectric nanoparticles. This is due to local fieldenhancement. In those embodiments that do in fact propose a metal outershell, there is an additional requirement as to the specific medium inwhich they must be used in order to properly function. Shell sizes have,in general, been relatively large, usually of the order of about 5 μm.In drug delivery applications, a smaller particle diameter is importantfor prolonged blood circulation and enhanced drug targeting to specificbody sites.

U.S. Pat. No. 6,479,146 describes a process for preparing coatedparticles and hollow shells by coating colloidal particles withalternating layers of oppositely charged nanoparticles andpolyelectrolytes, and optionally removing the colloidal cores. Theprocess involves the preparation of hollow silica microspheres vialayer-by-layer shell assembly on 640 nm diameter polystyrene latexparticles, followed by pyrolysis at 500° C. to decompose the polystyrenecore. The same assembly procedure was also used to preparesilica-containing shells on 3 μm diameter melamine-formaldehydeparticles, followed by acid dissolution of the core.

International Publication No. WO 2005/044224 describes a drug deliverysystem based on polymer nanoshells. In certain embodiments, thepolymeric nanoshells comprise one or more polymeric shells around ahollow core. In other embodiments, nanoshells are described which areuseful for the delivery of agents such as, for example, variousdiagnostic and therapeutic agents. The nanoshells disclosed arepreferably composed of biocompatible organic polymers which are mostpreferably biodegradable as well. The shell layers can comprisematerials such as gelatin, chitosan, dextrate sulphate, carboxymethylcellulose, sodium alginate, poly(styrene sulfonate) (PSS), poly(lysine),poly(acrylic acid), poly(dimethyldiallyl ammonium chloride) (PDDA) andpoly (allylamine hydrochloride) (PAH). However, the shells described arecomposed of an electrostatic interaction multilayer-based membrane. Thusnegative and positive charges are required on the particle, theelectrostatic nature of the surface induces interactions with proteinsand lipoproteins during the blood circulation. Such non-specificinteraction can decrease the nanoparticle lifetime.

Hu et al (Polymer, Vol. 46, Issue 26, 2005 pg. 12703-12710) describeformation of hollow polymeric nanospheres based on a core-template-freeroute, and the effects of polymerization concentration, shellcross-linking, pH, salt concentration and temperature on the size andstability of hollow polymeric nanospheres. The hollow structure ofpolymeric nanospheres is spontaneously formed by polymerization ofacrylic acid monomers inside the chitosan-acrylic acid assemblies. Thesize of the hollow nanospheres can be manipulated by changing pH, saltconcentration and temperature.

Li et al (Colloid & Polymer Science, Vol. 286, 6-7, pg. 819-825)describe biodegradable chitosan hollow microspheres where are preparedusing uniform sulfonated polystyrene (PS) particles as templates. Thechitosan was adsorbed onto the surface of the sulfonated polystyrenetemplates through the electrostatic interaction between the sulfonicacid groups on the templates and the amino groups on the chitosan andcrosslinked using glutaraldehyde. The controlled release behavior of thechitosan hollow microspheres was also primarily investigated aftertemplate removal.

The limited success of current pharmaceutical therapies is due to theabsence of an innovative drug delivery system which can increase thesafety and efficacy levels but also improve the overall performance ofthe therapeutic molecule. Stability and degradability control are twocritical aspects that must be developed to facilitate delivery.Controlling these parameters simultaneously is a greater challenge, andmost of the current vectors such as liposomes, microparticles andmicroemulsions do not support this, thus limiting their applications.Moreover, another major disadvantage of synthetic vectors is their lowin vivo efficiency. This is a consequence of their poor targetingability and their short lifetime due to the presence of surface positivecharge or the inherently low stability of their shells (liposomes).These factors lead to the degradation of the supramolecular structureand removal by macrophages before the vector arrives at the target cell.To circumvent these problems, hollow spheres appear to be a promisingstrategy. Recent interest in hollow spherical structures can beattributed to their unusual properties (chemical, mechanical andoptical) which suggest a wide range of applications. These structureshave potential utility in encapsulation and controlled release ofvarious biomolecules such as genes, peptides and drugs in clinicalapplications. Control of the structural characteristics of the hollowcarrier such as shell thickness, surface charge, pore size andmechanical strength is essential to achieving the aim of realising anideal encapsulation system.

Clearly, there is a need therefore to develop the next generation ofhollow nanospheres that are more robust, biocompatible and areresponsive to their environment, thus triggering the smart release ofthe biomolecule after delivery to the target. It is desirable that thesepolymeric systems would be biocompatible, capable of carrying a highpayload, capable of acting as a reservoir and could be programmed torespond to temperature and the like and be modified to target drugdelivery to a specific site.

It is thus an object of the present invention to provide improvedbiodegradable hollow mono-dispersed nanospheres which are adaptable totarget specific sites for use as carriers of and to allow targeted andcontrolled delivery of biomolecules and therapeutic agents and the likeor for use in other nanosphere applications.

It is a further object of the invention to provide biodegradablenanoshells which are adaptable to target specific sites, therebyallowing targeted and controlled delivery of biomolecules andtherapeutic agents and the like.

SUMMARY OF THE INVENTION

Accordingly, the invention provides a polymeric hollow nanosphere forrelease of an agent, wherein said nanosphere comprises at least onebiodegradable polymer, characterised in that said at least one polymeris cross-linked. Thus, the nanospheres of the invention comprises atleast one biodegradable polymer which is crosslinked with a crosslinkingagent.

In particular, the invention provides hollow biodegradable nanosphereswhich are specifically designed to allow for higher pay load capacityand which can act as a local reservoir with a controlled release profileand provide a sustained delivery of the therapeutic agent to the targetsite over time. These hollow nanospheres with a higher pay load capacitycan target drug or agent delivery to a specific site.

Cross-linking preferably arises from covalent linkages between thepolymer and the cross-linking agent.

In one aspect there is provided a polymeric hollow nanosphere forrelease of an agent,

wherein said hollow nanosphere comprises at least one natural orsynthetic biodegradable polymer, selected from the group consisting of:collagen, elastin, chitosan, hyaluronan, alginate, polyesters, PEG-basedpolymers, dendritic or hyperbranched polymers and combinations thereof,

wherein said at least one natural or synthetic biodegradable polymer iscross-linked by a cross-linking agent selected from at least one of thegroup consisting of a dendrimer, hyper-branched dendritic polymer and alinear polymeric system;

characterised in that the molar ratio of reactive —COOH or —NH₂ groupsin said cross-linker to reactive groups —COOH or —NH₂ in said polymer isin the range 50:1 to 1:50.

The invention thus relates to the design of hollow biodegradablenanospheres which are specifically designed to allow for higher pay loadcapacity and which can act as a local reservoir with a controlledrelease profile and provide a sustained delivery of the therapeuticagent to the target site over time. These hollow nanospheres with ahigher pay load capacity can target drug or agent delivery to a specificsite.

The skilled person will appreciate that the ratio of cross-linking agentto polymer can be varied to tailor the physical properties of thenanosphere. The choice of cross-linking agent selected for cross-linkingthe polymeric nanosphere will also affect the physical properties of thenanosphere.

In all embodiment described herein, by the phrase “the ratio ofcross-linker to polymer” it is meant that the ratio of reactive —COOH or—NH₂ groups in said dendrimer cross-linker to reactive groups —COOH or—NH₂ in said polymer. This ratio is a molar ratio.

For all disclosed embodiments it is preferred, the molar ratio ofreactive —COOH or —NH₂ groups in said dendrimer cross-linker to reactivegroups —COOH or —NH₂ in said polymer is in the range of 10:1 to 1:10 andmore particularly preferred 5:1 to 1:5. Preferably the inventionprovides a polymeric nanosphere wherein the molar ratio of cross-linkerreactive groups to polymer reactive groups is in the range of 5:1 to1:5. In the most preferred embodiment of the invention, the ratio ofcross-linker to polymer is 1:2. The inventors have found that a ratio of1:2 cross-linker to polymer produces well defined nanospheres. Inanother preferred embodiment, the ratio of cross-linker to polymer maybe 1.5:2. This ratio is a molar ratio. This ratio is a molar ratio.

The biodegradable polymer may be selected from the group consisting of:collagen, elastin, chitosan, hyaluronan, alginate, polyamidoamine(PAMAM), poly-l-lysine (PLL), polyethyleneimine (PEI), polyglutamic acid(PGA), poly (ethylene glycol) methacrylate (PEGMA) and poly (propyleneglycol) methacrylate (PPGMA), (meth) acrylic acid monomers (e.g.methacrylic acid and acrylic acid) or NHS monomers (e.g. acrylic acidN-hydroxysuccinimide ester) with multi-functional vinyl monomers (e.g.PEG dimethacrylate), and polycarboxylic acid-polyethyleneglycol-polycarboxylic acid polymers such as APEGA, and combinationsthereof. In a preferred embodiment peptides amphiphiles such as thosewell known in the art may be used. Peptide amphiphiles includepoly(L-lysine), TMA[-Ala-TRIS[(Gly-Pro-Nleu)₆-OMe]₃]₃, Ala periphery[(β-Ala₈-Orn₄-Orn₂-Orn-NH—CH₂—CH₂—)₂,[(H-Gly-Pro₅)₂-Amp]₂-(Gly-Pro₅)₂-Amp-CONH₂, and Proline-rich MAPs.

In a preferred embodiment, the biodegradable polymer may comprise acombination of polymers selected from the group consisting of: chitosanand polyglutamic acid; PAMAM and hyaluronan; and chitosan and collagen.

Natural biodegradable polymers are preferred and include biodegradablepolymers such as collagen, elastin, chitosan, hyaluronan or alginate andcombinations thereof. The skilled person will appreciate that elastinincludes elastin like polypeptide (ELP) or pre-elastin such astropoelastin can also be used. Mixtures of at least two natural polymersare particularly preferred.

Preferred synthetic biodegradable polymer include polyesters, PEG-basedpolymers, dendritic or hyperbranched polymers and combinations thereof.

Preferably, the cross-linking agent is selected from the groupconsisting of dendrimers or dendritic polymeric systems, glutaraldehyde,carbodiimides, genepin, transglutaminase, sulfonates, including methylsulfonate and trifluoromethyl sulfonate, and malemide. Glutaraldehyde isless preferred as it is toxic. The skilled person will appreciate thatcross linking promoters can be used to induce/assist crosslinking.However, dendrimers or dendritic polymeric systems are the mostpreferred cross-linking agents for the natural polymeric hollownanospheres of the invention.

The most suitable cross-linking agents include dendrimers or dendriticpolymeric systems. Dendrimers are spherical, highly branched polymershaving specific functionalised surface chemistries. Dendritic polymericsystems include dendritic architectures such as dendrimers, dendronized,hyperbranched and brush-polymers. The area of dendritic molecules can beroughly divided into the low molecular weight and the high molecularweight species. Dendrimers of different generations can be usedaccording to the needs of the particular application. For example,dendrimer or dendritic polymeric systems of different generations, e.g,G₁-G₁₀ or half generations may be used.

Preferably, the dendrimer is a PPEGP based dendrimer. The dendrimer cancomprise a tricarboxylic acid monomer core (such as an aconitic acidcore) and polyol branching monomer (such as PEG). Thus, the dendrimermay suitably comprise an aconitic acid (core) and PEG (surface) baseddendrimer such as aconitic acid-polyethylene glycol-aconitic acid baseddendrimer (APEGA). More preferably, the dendrimer may comprise apre-activated carboxylic acid functionalised polyether dendrimer, suchas a pre-activated carboxylic acid functionalised APEGA dendrimer.

The degradation products of the nanospheres according to the inventionare biocompatible and absorbable. Natural polymers typically degrade inthe body to into low molecular weight peptides, oligosaccharides andvery low molecular weight hyaloronan.

Advantageously, the rate of degradation and thus lifetime, of thenanosphere can be tailored by cross-linking the polymer matrix withdifferent cross-linkers (co-polymers) and by modulating thecross-linking ratio. More strongly cross-linked hollow nanospheres willbe more resistant to degradation and will take longer to degrade thanless cross-linked hollow nanospheres. The skilled person will appreciatethat the ratio of cross-linking agent to polymer can be varied to tailorthe physical properties of the nanoshell. The choice of cross-linkingagent selected for cross-linking the polymeric nanoshell or nanospherewill also affect the physical properties of the nanoshell. The skilledperson will appreciate that the ratio of cross-linker to polymer can beselected according to the desired end properties of the nanosphere, forexample degree of biodegradability, rigidity, and biological properties.

Furthermore, the nanoshells and nanospheres of the invention illustratethat cross-linking of the polymers forming the nanosphere improves thestability and mechanical integrity of the nanosphere. Furthermore, thedegree of cross-linking can affect various physical properties of thenanosphere such as the permeability of the sphere wall, the rate ofdegradation of the sphere, and the rate of release of agentsencapsulated within the sphere, for example.

Dendrimers or dendritic polymeric systems are the preferredcross-linking agents for the natural polymeric hollow nanospheres of theinvention.

The nanospheres as described herein are superior to existing nanospheresinsofar as they can be developed with a neutral surface charge ratio.Neutral surfaces are preferred over ionic surfaces since non-specificinteractions are decreased and thus the nanosphere lifetime isaugmented. Furthermore, covalent surface linkages are preferred overionic surfaces since nanospheres comprising covalent surfaces mean thatthe nanospheres of the present invention can be used at neutral pH or inphysiological serum.

The dendrimer may be selected from the group consisting ofpolyamidoamine (PAMAM), polypropyleneimine (PPI), polyarylether (PAE),polyethyleneimine (PEI), poly-l-lysine (PLL), polyacrylicacid-(polyethylene glycol)-polycarboxylic acid (PPEGP); polycarboxylicacid-(polyethylene glycol)-polycarboxylic acid (PPEGP); or frompoly(ethylene glycol)methacrylate (PEGMA) and poly(propyleneglycol)methacrylate (PPGMA) mixtures, in ratios ranging from 70:30 to25:75.

The dendritic polymeric systems synthesized from the combinations of thelatter two polymers with tailored functional groups (carboxylic or NHSgroups) can be designed and synthesized via the controlled/living freeradical copolymerisations (ATRP or RAFT) of conventional (meth) acrylicacid monomers (e.g. methacrylic acid and acrylic acid) or NHS monomers(e.g. acrylic acid N-hydroxysuccinimide ester) with multi-functionalvinyl monomers (e.g. PEG dimethacrylate). These resultant carboxylic orNHS functionalized dendritic polymeric systems can be conjugated orcross-linked with peptides, proteins and chitosan via their aminefunctional groups, for the tethering of drug/growth factors.

Peptide-based dendrimers such as poly(L-lysine),TMA[-Ala-TRIS[(Gly-Pro-Nleu)₆-OMe]₃]₃ improve the exposure of thefunctionalities to the surrounding environment and mimic thearchitectures of biological structures which have naturally evolved tofacilitate specific bio-interactions. They also exhibit desirablebiological activities and facilitate the synthesis of highly controlledconstructs of consistent size, architecture and composition.Multi-antigenic peptides form a dendrimeric structure with variousexposed functionalities. Nanostructured peptide dendrimers based ondifferent amino acids, including lysine, exhibit promising vaccine,antiviral and antibacterial properties. Exploiting the inherent propertyof several surfactant-like peptides which undergo self-assembly enablesthe formation of nanotubes and nanovesicles having an average diameterof 30-50 nm with a helical twist.

In a particularly preferred embodiment, there is provided a polymerichollow nanosphere for release of an agent, wherein said nanospherecomprises at least one natural biodegradable polymer, selected from thegroup consisting of: collagen, elastin, chitosan, hyaluronan andalginate, wherein said at least one polymer is cross-linked by across-linker agent selected from the group consisting of: a dendrimer ora dendritic polymeric system selected from the group consisting of:aconitic acid-polyethylene glycol-aconitic acid based dendrimer (APEGA),polyamidoamine (PAMAM), polypropyleneimine (PPI), polyarylether (PAE),polyethyleneimine (PEI), poly-l-lysine (PLL), polyacrylicacid-(polyethylene glycol)-polycarboxylic acid (PPEGP); polycarboxylicacid-(polyethylene glycol)-polycarboxylic acid (PPEGP); or frompoly(ethylene glycol)methacrylate (PEGMA) and poly(propyleneglycol)methacrylate (PPGMA) mixtures, in ratios ranging from 70:30 to 25:75characterised in that the molar ratio of reactive —COOH or —NH₂ groupsin said dendrimer cross-linker to reactive groups —COOH or —NH₂ in saidpolymer is in the range 50:1 to 1:50. More preferably, the molar ratioof reactive —COOH or —NH₂ groups in said dendrimer cross-linker toreactive groups —COOH or —NH₂ in said polymer is in the range 5:1 to1:5.

The polymeric coating of the nanosphere may be cross-linked withdendrimer using EDC/NHS coupling. The use of a dendrimer using EDC/NHScoupling makes more functional groups available for surface modificationof the shells such as for tethering imaging agents or specific sitereceptors, for example. A pre-activated carboxylic acid functionalisedpolyether dendrimer, such as a pre-activated carboxylic acidfunctionalised APEGA dendrimer can also be used, since such dendrimersare activated for facilitating polymer cross-linking without the needfor use of carbodiimide chemistry. Aconitic acid-polyethyleneglycol-aconitic acid based dendrimer (APEGA) is particularly preferred.

Biodegradable dendrimers such as poly(glycerol-succinic acid) dendrimer(PGLSA), poly(2,2-bis(hydroxymethyl)propionic acid) providebiodegradable crosslinking polymer without production of toxic residuesand allow programming of cross-linked spaces in the polymer in question.This is advantageous over use of EDC/NHS coupling, since this can oftenresult in production of zero-length cross links within the polymer.

In accordance with the present invention, nanospheres of specific sizesare prepared for their absorbance by specific cells in the body. Theinvention provides mono-dispersed biodegradable polymer basednanospheres of a range of dimensions.

The nanospheres of the invention may be sized in the range 1 nm to 5000nm. Preferably, the nanosphere has a size in the range from 10 nm to 50nm, more preferable 50 nm to 100 nm, more preferable still from 100 nmto 200 nm, from 200 nm to 500 nm, more preferably from 0.1 to 5 μm (100nm to 5000 nm). Further preferably, the nanosphere has a size in therange of 0.1 to 2 μm (100 nm to 2000 nm). The size of the nanosphere isof particular importance for controlled delivery of agents to targetcells in the body. The nanosphere will be smaller in size than thetarget cell, which has an average size of 50 μm.

In a preferred embodiment, the polymeric chitosan nanospheres of thepresent invention can be cross-linked directly with carboxylic acidfunctionalised dendritic polymeric systems or with NHS-functionaliseddendrimers or dendritic polymeric systems. The polystyrene core templatecan be removed from the nanosphere template by dissolution using THF.The preferred chitosan nanospheres can be fabricated and functionalizedaccording to the methodology described herein.

Preferably, the nanospheres according to the present invention providefor controlled release of an agent at a target site. Further preferably,the nanospheres of the present invention provide for controlled,targeted delivery of an agent or agents to a site.

The controlled delivery is one of the most important goals for syntheticgene delivery. It has been proved that moieties such as polysaccharides,antibodies and peptides, for example increase the targeting of specificcells (e.g. mannose to macrophages; galactose to hepatocytes; folic acidto cystic fibrosis cells, etc.).

The polymeric nanospheres according to the invention comprise a hollowcavity. The hollow cavity within the shell can be used to encapsulateagents that require delivery to a target cell. The agent may be selectedfrom the group consisting of biomolecules, therapeutic agents or imagingagents.

Suitable agents include but are not limited to pDNA, polyplexes, growthfactors, peptides, viral and non-viral vectors (pDNA), doxorubicin,genes, hormones, enzymes, FITC, tryptophan, rhodamine,4′,6-diamidino-2-phenylindole (DAPI) and TOPRO3, for example. Otheragents include fluorescein and it's derivatives, red dyes, green dyessuch as AlexaFlor; and fluorescent proteins such as GFP/eGFP, YFP, andchemicals, APIs, drugs and pro-drugs.

Encapsulation of the therapeutic agent protects it from the outsideenvironment, thereby, increasing the half-life of the agent.Encapsulation also protects the therapeutic agent from non-specific siteinteraction during penetration. Thus, the biomolecule is protectedduring the journey of the device to the target site.

In one embodiment the nanospheres according to the invention can be usedfor molecular imaging by impregnating the shells with imaging agents.Accordingly, the nanosphere according to the invention may furthercomprise an imaging agent. The imaging agent may be encapsulated in theshell. In an alternative embodiment an imaging agent may be attached tothe exterior surface of the nanosphere. The imaging agent may beselected from the group consisting of FITC, tryptophan, rhodamine,4′,6-diamidino-2-phenylindole (DAPI) and TOPRO3. Other agents includefluorescein and it's derivatives, red dyes, green dyes such asAlexaFlor; and fluorescent proteinssuch as GFP/eGFP, YFP, and chemicals,APIs, drugs and pro-drugs.

In an alternative embodiment, the nanosphere according to the inventionmay further comprise a homing mechanism. The presence of a homingmechanism facilitates targeted delivery of an agent by the nanosphere.The homing mechanism may be selected from the group consisting of asaccharide including lactose, galactose and manose; folic acid, anantibody fragment or a peptide sequence.

In a further embodiment of the current invention the surface of thepolymer shell may be tagged with a fluorescent marker for traceabilityand a homing mechanism for delivery to specific target sites. Thefunctional groups on the outer surface of the nanosphere may be suitablylabelled by treating said nanospheres with fluorescein isothiocyanate(FITC), 8-anilino-1-naphthalenesulfonic acid (ANS) or any otherfluorophores, for example.

Dendrimeric cross-linking systems (of the type described herein) can beused to conjugate the imaging, therapeutic and homing tags. Dendrimericsystems are used to increase the number of functional groups which canbe used to conjugate any tags, imaging or therapeutic moieties.Excessive functional groups of the polymer shells can be modified withbiocompatible molecules such as poly (ethylene glycol), to suppress theimmunity of the device.

In one embodiment, the polymeric nanosphere according to the inventioncomprises one or more polymeric layers. The polymeric layers maycomprise at least one polymer selected from the group consisting ofpolyglutamic acid, hyaluronan, alginate, PLGA, poly(caprolactone) andpoly(d,l-lactide). The skilled person will appreciate that othersuitable polymers could also be used. The invention therefore alsoprovides multilayer polymeric nanospheres.

The invention also provides a process for the preparation ofbiodegradable hollow polymeric nanospheres comprising the steps of:

-   -   (i) providing a template comprising polymeric nanoparticles;    -   (ii) treating said polymeric nanoparticles with a        functionalising group to produce functionalised nanoparticles;    -   (iii) treating said functionalised nanoparticles with a solution        of one or more natural polymers, and agitating to form a        polymeric coating on said nanoparticles    -   (iv) cross-linking said coating; and    -   (v) removing the template by treating said particles with a        solvent.

In a particularly preferred embodiment, there is provided a process forthe preparation of a natural or synthetic biodegradable polymeric hollownanosphere comprising the steps of:

-   -   (i) providing a template comprising polymeric polystyrene beads,        mesoporous silica or diatomaceous silica;    -   (ii) treating said template with a functionalising group to        produce a functionalised template,    -   (iii) treating said functionalised template with a solution of        one or more natural or synthetic biodegradable polymers selected        from the group consisting of: collagen, elastin, chitosan,        hyaluronan, alginate, polyesters, PEG-based polymers, dendritic        or hyperbranched polymers, and combinations thereof, and        agitating to form a polymeric coating on said template;    -   (iv) cross-linking said coating with a cross-linking agent        selected from the group comprising a dendrimer, a hyper-branched        dendritic polymer and linear polymeric system, and combinations        thereof, wherein the molar ratio of reactive —COOH or —NH₂        groups in said dendrimer cross-linker to reactive groups —COOH        or —NH₂ in said polymer is in the range 50:1 to 1:50; and    -   (v) removing the template by treating said template with a        solvent.

Thus, the current invention provides for a method for producing a hollownanosphere made of a biodegradable polymeric sphere. The use of anatural or synthetic biodegradable polymer will reduce the risk ofchocking and compatibility issues. Natural biodegradable polymersdisclosed herein are preferred.

The templates may comprise beads such as polystyrene beads, includingsubstituted derivatives thereof, such as polystyrene sulfonated (PSS),carboxylated or aminated polystyrene beads; silica beads; polyesterbeads including polycaprolactone (PCL), polylactide (PLLA), polyethyleneterephtalate (PET), polycarbonate, polybutyrate; polyamides includingpolyacrylamide (PAA), polyamidoamide (PAMAM); acrylic polymers includingpolyacrylate, polymethacrylate (PMA), polymethylmethacrylate; poly(ethylene glycol) methacrylate (PEGMA) and poly(propyleneglycol)methacrylate (PPGMA), (meth)acrylic acid monomers (e.g.methacrylic acid and acrylic acid) or NHS monomers (e.g. acrylic acidN-hydroxysuccinimide ester), multi-functional vinyl monomers (e.g. PEGdimethacrylate) and cationic, anionic or amphiphilic polymers includingPEI, PLL, PEG, PGA, PLGA, hyaluronan, chitosan and collagen; carbonnanotubes and metallic beads such as gold beads. The skilled person willappreciate that other suitable polymeric beads could also be used. Inparticular, the skilled person will appreciate that any suitable ionicparticles or complexes can be used as a template to prepare thenanospheres according to the invention.

Preferably, the beads selected for use as templates in accordance withthe present invention comprise beads of a size suitable for use inproducing a desired size of nanospheres. Preferably, the beads templatescomprise beads of a size in the range 0.05 to 5 μm.

In the preferred embodiment, the size of the nanospheres can becontrolled by using polystyrene beads having a size in the range 0.05 to5 μm as template, for example.

The skilled person will appreciate that using specific reactionconditions can produce specific sizes of nanosphere. For example, in thecase of the polymerization of styrene, as described in the examples, thequantity of initiator (such as AIBN, BPO) or the solvent ratio(ethanol/water) used during polymerisation induces the production ofbeads having a size in the range from 0.05 to 5 μm in diameter.

In one embodiment, the template may be in the form of a dendrimer ordendritic polymeric system template. Dendrimers or dendritic polymericsystems are particularly suited to use as nanoshell and nanospheretemplates, since dendrimers or dendritic polymeric systems of a widerange of sizes are accessible. For example, nanosphere size variationscan be achieved by using dendrimers of different generations, e.g,G₁-G₁₀ or half generations thereof etc., as template. It will beappreciated that different shaped dendrimers or dendritic polymericsystems, e.g., spherical, globular or bowtie dendrimer templates mayresult in different shaped nanoshell and nanosphere.

Another advantage from use of dendrimer or dendritic polymeric systemtemplates stems from the fact that the dendrimer or dendritic polymericsystem template may have built in functionality. This is useful forfacilitating polymer bonding around the dendrimer or dendritic polymericsystem template.

The functionalising group may be selected from the group consisting ofsulphate, a carboxyl and amine groups.

Preferably the functionalising group comprises sulphate. The term“functionalised nanospheres” as used herein means nanospheres whosesurface has been modified by the attachment of a functional group. Themeaning of the term “functional group” will be known to the personskilled in the art. The term “functional group” refers to specificgroups of atoms which form part of molecules and which are responsiblefor the characteristic chemical behaviour of those molecules.

Preferably, the nanoparticles or polymeric nanosphere template may beselected from the group consisting of sulfonated polystyrene beads,polymethylmethacrylate beads, silica beads and functionalised dendrimersor dendritic polymeric bead type systems.

In a preferred embodiment, the polymeric nanoshell and nanospherescomprise sulfonated polystyrene beads. Sulfonated polystyrene beads canreadily be produced on a large scale. They have a low reactivity in theprocess of formation of the nanospheres and can be readily removedfollowing formation of the nanosphere.

The natural polymer for the polymeric solution may be selected from thegroup consisting of a protein, a polysaccharide, a cationic polymer,dendrimer and a polyacid.

Suitably, the polymeric solution comprises a polymer selected from thegroup consisting of collagen, gelatin, elastin, chitosan, hyaluronicacid, alginate, polyamidoamine (PAMAM), poly-l-lysine (PLL),polyethyleneimine (PEI), polyglutamic acid (PGA), poly (ethylene glycol)methacrylate (PEGMA) and poly (propylene glycol) methacrylate (PPGMA),(meth) acrylic acid monomers (e.g. methacrylic acid and acrylic acid) orNHS monomers (e.g. acrylic acid N-hydroxysuccinimide ester),multi-functional vinyl monomers (e.g. PEG dimethacrylate) and apolycarboxylic acid-polyethylene glycol-polycarboxylic acid (PPEGP)based dendrimer such as APEGA and combinations thereof. In a preferredembodiment, the APEGA dendrimer has pre-activated carboxylic acidsurface functionality.

Suitably, the preferred polymeric solution comprises a naturalbiodegradable polymer selected from the group consisting of collagen,elastin, chitosan, hyaluronic acid and alginate.

In one aspect, the polymeric solution may comprise natural polymers inthe form of a dendrimer polymer. The size of such dendrimers used as thematerial to build up the nanosphere layer can be varied according toneed, since any particular number or combination of dendrimergenerations or half generations can be used as the natural polymersubstance. It will be appreciated that the size of dendrimer which maybe required in this role will depend on a number of factors such as sizeof nanoshell and nanosphere etc.

In a preferred embodiment, the sulfonated polystyrene beads are suitablycoated by dispersing them in a polymeric solution.

Different families of (multi)polymer based nanospheres may be producedin accordance with the present invention. The polymeric solution mayfurther comprise a combination of two or more polymers. The use of acombination of two or more polymers allows the production of multipolymer nanospheres. The properties of such multi polymer nanospheressuch as water solubility, surface charge ratio and porosity can beadjusted using a well defined combination of polymers.

Preferably the polymeric solution comprises a combination of polymersselected from the group consisting of chitosan/PGA; PAMAM/hyaluronan andchitosan/collagen. The skilled person will appreciate that othersuitable combinations of polymers could also be used.

In a preferred embodiment of the process according to the invention, thepolymeric coating is crosslinked using a ratio of cross-linker topolymer of 50:1.

In a preferred embodiment of the process according to the invention, thepolymeric coating is crosslinked using a ratio of cross-linker topolymer of 1:2.

In an alternative embodiment of the process according to the invention,the polymeric coating is crosslinked using a ratio of cross-linker topolymer of 1.5:1.

Preferably, the polymeric coating is cross-linked with a cross-linkerselected from the group consisting of dendrimers or dendritic polymericsystems, glutaraldehyde, carbodiimides, genepin, transglutaminase,sulfonates including methyl sulfonate or trifluoromethyl sulfonate, forexample, and malemide.

Further preferably, the solid particles are cross-linked with adendrimer or dendritic polymeric system. The dendrimer may be selectedfrom the group consisting of polyamidoamine (PAMAM), polypropyleneimine,polyarylether and polyethyleneimine (PEI), poly-l-lysine (PLL), poly(ethylene glycol) methacrylate (PEGMA), poly (propylene glycol)methacrylate (PPGMA), polyacrylic acid and a polycarboxylic acid-polyethylene glycol-polycarboxylic acid based dendrimer such as APEGA,and combinations thereof. In a preferred embodiment, the APEGA dendrimerhas pre-activated carboxylic acid surface functionality.

Thus in one particular embodiment, it is possible to use a dendrimer asa nanosphere template, as the nanoshell and nanosphere boundary orsurface polymer and as crosslinker of said surface polymer to addmechanical strength and integrity to the nanosphere. It will beappreciated that the same or different types of dendrimer or dendriticpolymeric systems and dendrimer generations can be used for each ofthese distinct roles.

Furthermore, the process according to the invention may further comprisethe step of treating the polymer coated beads with a second polymericsolution comprising a polymer selected from the group consisting ofpolyglutamic acid, hyaluronic acid, alginate, PLGA, poly(caprolactone)and poly(d,l-lactide) to produce multilayer polymeric particles. Theskilled person will appreciate that other suitable polymers could alsobe used.

The properties of such multi polymer nanoshells or nanospheres such aswater solubility, surface charge ratio and porosity can be adjustedusing a well defined combination of polymers.

Suitably, the process according to the invention further comprisesremoval of the templates by treating the particles with a solutionsuitable for dissolving the template. Typically acid solutions may beused to dissolve the template. THF is a preferred solvent for suchdissolving solutions. For example, 1% acetic acid solution withTHF:water (80:20 volume) may be used, prior to centrifugation to producea nanosphere.

Preferably, the process according to the invention further comprises thestep of encapsulating a biomolecule, therapeutic or imaging agent in thenanospheres. The nanospheres are sterilised prior to encapsulating theagent(s).

Preferably the therapeutic agent is encapsulated in said nanospheres bymeans of physical diffusion. The therapeutic agent is preferablyencapsulated in the nanosphere by the dispersion of the nanospheres in asolution of specific therapeutic agent to allow diffusion of thetherapeutic agent inside the cavity of the nanosphere.

Alternatively, the therapeutic agent is encapsulated in said nanospheresby means of emulsion. Molecular imaging agents may also be impregnatedinto the hollow cavity of the sphere by physical diffusion or emulsion.

Preferably, the process according to the invention further comprises thestep of incorporating a homing mechanism on the nanosphere surface.

The invention provides a vehicle for delivering an agent to a site,comprising a nanosphere obtained by the process as described herein. Theagent may be selected from the group consisting of biomolecules,therapeutic agents and imaging agents, for example.

The current invention therefore provides nanospheres for targeteddelivery of an agent or agents to target cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an SEM of polystyrene nanoparticles;

FIG. 2 shows an SEM of uncross-linked chitosan nanoshells;

FIG. 3 shows SEM analysis of nanoshells from batches 1 to 3 fromTable 1. SEM analysis (left hand side) of nanoshells from batch no 1(A), batch no 2 (B) and batch no 3 (C);

FIG. 4 shows TEM analysis of nanoshells from batches 1 to 3 fromTable 1. TEM analysis of nanoshells from batch no 1 (A), batch no 2 (B)and batch no 3 (C) of Table 1; and

FIG. 5 shows the DNA encapsulation ratio comparison between chitosannanoshells crosslinked with glutaraldehyde and chitosan/PGA nanoshells;

FIG. 6 shows that conjugated FITC-PAMAM G1 does not migrate from theorigin using TLC;

FIG. 7 shows mass spectra of FITC labelled PAMAM in negative ion mode byflow injection analysis. Mass spectra of FITC labelled PAMAM in negativeion mode. Analysis carried out by flow injection analysis with anAgilent ion trap (6330 model);

FIG. 8 shows confocal micrographs of 3T3 fibroblasts stained with RP(red) through RP filter (left hand side) and FITC filter (right handside). Confocal micrographs of 3T3 fibroblasts stained with RP (red)through RP filter (left hand side);

FIG. 9 shows confocal micrograph of 3T3 cells stained with RP andincubated with FITC-PAMAM complex. Confocal micrograph of 3T3 cellsstained with RP and incubated with FITC-PAMAM complex;

FIG. 10 shows SEM (A) and TEM (B) images of polystyrene beads. SEM (A)and TEM (B) images of polystyrene beads;

FIG. 11 shows FTIR spectra of polystyrene and sulfonated polystyrenebeads. FTIR spectra of polystyrene and sulfonated polystyrene beads;

FIG. 12 shows SEM images of sulfonated polystyrene beads coated withvarying concentrations of chitosan and sulfonated polystyrene beadscoated by chitosan cross linked with PGA. SEM images of sulfonatedpolystyrene beads coated with 50 mg of chitosan (A), 125 mg (B), 250 mg(C) and 375 mg (D);

FIG. 13 shows FTIR spectra of polystyrene and shells. The tagged peaksare characteristic of polystyrene beads. FTIR spectra of polystyrene(blue) and shells (purple). The tagged peaks are characteristic ofpolystyrene beads;

FIG. 14 shows effect of the protocol (1 or 2) on the encapsulation ratioand confocal micrograph of ethidium bromide labeled pDNA. Effect of theprotocol (1 or 2) on the encapsulation ratio and confocal micrograph ofEthidium bromide labeled pDNA (red A) encapsulated into FITC labeledshells (green B). The resulting complexes appear yellow (C);

FIG. 15 shows fluorescence microscopic images of HUVEC with differentsizes of negatively charged hollow spheres at 12 hours. Fluorescencemicroscopic images of HUVEC with different sizes of negatively chargedhollow spheres at 12 hr (A) 200 nm (B) 400 nm (C) 600 nm hollow sphereswith HUVEC;

FIG. 16 shows confocal micrographs of Ethidium bromide labeledencapsulated pDNA. (A) confocal micrograph of Ethidium bromide labeledpDNA (red A) encapsulated into FITC labeled spheres (green B). Theresulting complexes appear yellow (C) (scale bar: 2 μm), (B) confocalmicrograph of rhodamine (red) stained endothelial cells (HUVEC)incubated 12H with 500 nm FITC labelled spheres (green) (scale bar: 50μm);

FIG. 17 shows electrophoresis gel with ladder (1 kb) of encapsulatedpDNA using protocol 1 and protocol 2. Electrophoresis gel (0.9% agarose)with ladder (1 kb) (1), pDNA (2), Shells (3), pDNA encapsulated intoshell by using protocol 1 and after wash (4), pDNA encapsulated intoshell by using protocol 1 and before wash (5), protocol 1 washed phase(6) and protocol 2 (7);

FIG. 18 shows release of protocol 1 encapsulated pDNA in FBScomplemented media and in presence of protease (enzyme) over a 72-hourtime period. Release of protocol 1 encapsulated pDNA in FBS complementedmedia (DMEM 10% FBS) and in presence of protease (enzyme) over a 72-hourtime period;

FIG. 19 shows electrophoresis gel after enzymatic release with Ladder (1kb) up to 72 hours. Electrophoresis gel (0.9% agarose) after enzymaticrelease with Ladder (1 kb) (1), 6 H incubation time (2), 72 H incubationtime (3), native pDNA (4);

FIG. 20 shows electrophoresis gel (0.9% agarose) after DNase1 exposurewith Ladder (1 kb) of native pDNA, and Shell/pDNA complex with andwithout exposure to DNase1. Electrophoresis gel (0.9% agarose) afterDNase1 exposure with Ladder (1 kb) (1), native pDNA (2), pDNA exposed toDNase1 (3), Shell/pDNA complex (4), Shell/pDNA complex exposed to DNase1(5), extracted Shell/pDNA complex (6) and extracted Shell/pDNA complexexposed to DNase1 (7);

FIG. 21 shows fluorescent microscope micrographs of HUVEC cell stainedwith rhodamine phalloidin incubated up to 48 hours. Fluorescentmicroscope micrographs of HUVEC cell stained with rhodamine phalloidin(red) incubated 6 H(A) and 48 H (B) with FITC labelled spheres (green),scale bare (50 μm);

FIG. 22 shows the charge effect of the nanospheres (nanoshells) onhaemolysis;

FIG. 23 shows the effect of size of the nanospheres (nanoshells) onhaemolysis;

FIG. 24 shows the charge effect of the nanospheres (nanoshells) onplatelet activation determined by the amount of sP-selectin in solution;and

FIG. 25 shows the size effect of the nanospheres (nanoshells) onplatelet activation determined by the amount of Sp-selection insolution;

FIG. 26 shows Zeta potential analysis of 100, 300, 500 and 1000 nmhollow spheres in mV;

FIG. 27 shows TNBSA analysis of mTGase cross-linked hollow nanospheresrelative to controls. TNBSA analysis of cross-linking of hollownanospheres with various amounts of mTGase enzyme units havingglutaraldehyde as a positive control. Statistical significance wasdetermined by one way ANOVA (n=9, p<0.05);

FIG. 28 shows PicoGreen® assay and zeta potential analysis of polyplexloading inside hollow sphere. PicoGreen® assay and zeta potentialanalysis of polyplex loading inside hollow sphere. (A) PicoGreen® assayshowing difference of loading percentage by the direct quantification ofpolyplex and treating the polyplex with PGA for quantification (B) zetapotential analysis of hollow spheres before loading (BL), after loading(AL) and after treatment with PGA to validate PGA method forquantification. Statistical significance was determined by one way ANOVAand student's t-test (n=3, p<0.05);

FIG. 29 shows PicoGreen® assay showing amount of polyplex loadingoutside spheres and inside the hollow spheres after treating the sphereswith PGA. PicoGreen® assay showing amount of polyplex loading outsidehollow spheres and inside the hollow spheres after treating the sphereswith PGA. Statistical significance was determined by one way ANOVA (n=3,p<0.05);

FIG. 30 shows a comparison of pDNA and polyplex loading behavior ofhollow spheres and solid spheres. pDNA and polyplex loading behavior ofhollow spheres (A) comparison of pDNA loading efficiency of hollowspheres and solid sphere, (B) pDNA loading efficiency of hollow spheresusing pDNA alone and polyplex, (C) loading efficiency of 1000 nm hollowspheres with varying ratios of polyplex to sphere, (D) loadingefficiency of all four different sizes of hollow spheres and (E) TEMimage of 1000 nm hollow sphere loaded with polyplex. All the data arerepresented as the mean±standard deviation (n=3). Statisticalsignificance difference was determined using one-way ANOVA and student'st-test. * indicates a statistically significant difference betweensamples with p<0.05;

FIG. 31 shows SDS-PAGE showing the gradual cross-linking of hollowsphere with increase in mTGase amounts. The gel was stained usingcoomassie blue;

FIG. 32 shows TEM micrograph of self assembled solid spherescross-linked with mTGase and 20% THF;

FIG. 33 shows PicoGreen® assay and agarose gel electrophoresis showingrelease of pDNA from the polyplex using polyglutamic acid. PicoGreen®assay and agarose gel electrophoresis showing release of pDNA from thepolyplex using polyglutamic acid. (A) PicoGreen® assay showing emissionvalues and (B) agarose gel of pDNA, polyplex and polyplex treated withPGA. Statistical significance was determined by one way ANOVA (n=3,p<0.05);

FIG. 34 shows release profile of hollow spheres. Release profile ofhollow spheres (A) cumulative release profile of pDNA/polyplex from allfour different sizes of hollow spheres at 37° C., (B) in vitro releasestudy of hollow spheres in the presence of 10 U/g of protease (pH 7.5),(C1) SEM image of untreated 1000 nm hollow spheres, (C2) degradinghollow spheres in the presence of protease after 72 hours;

FIG. 35 shows cell viability and transfection efficiency of GLP loadedhollow spheres. Cell viability and transfection efficiency of GLP loadedhollow spheres. PicoGreen® assays showing the cell viability of loadedhollow spheres of all four different sizes in (A) ADSCs and (B) HUVECSafter 48 hours. Gaussia luciferase assay for investigation oftransfection efficiency of all four different sizes polyplex loadedhollow spheres (C). All the data are represented as the mean±standarddeviation (n=3). Statistical difference was determined using one-wayANOVA. * indicates a statistically significant difference betweensamples with p<0.05;

FIG. 36 shows TEM images showing the internalization pathway of 500 nmhollow sphere loaded with polyplexes within ADSCs. TEM images showingthe internalization pathway of 500 nm hollow spheres loaded withpolyplexes within ADSCs. Hollow spheres were observed at differentlocations in the cell (A) attached to cell membrane, (B) cell membraneengulfing a sphere, (C) in early endosomes close to the cell membrane,(D) in late endosome or lysosome near to nucleus, (E) rupturing thelysosomal membrane and also spheres are degrading and (F) coming out ofthe lysosome by completely disrupting the lysosomal membrane. Cellmembrane, endosomes and lysosome have been represented as CM, E and Lrespectively in the figures;

FIG. 37 shows confocal micrographs of FITC labelled hollow spheres.Confocal micrographs of FITC labelled hollow spheres (A) 100 nm and (B)300 nm internalized into HUVECs and (C) 100 nm and (D) 300 nminternalized into HUASMCs. All images were taken after 24 hoursincubation with cells. Confocal microscopy of adipose derived stem cellsafter (D) 30 min (E) 2 h (F) 4 h (G) 24 h, incubation with nanospheres;

FIG. 38 shows TEM images illustrating 100 nm neutrally charged hollowspheres internalized into HUVECs and HUASMCs cells. TEM imagesillustrating 100 nm neutrally charged spheres internalized into (A)HUVECs inset shows the hollow spheres inside lysosome), (B) HUASMCs(inset shows hollow spheres inside lysosome) (C) Higher Magnificationimage of (A) showing endocytic internalization of hollow spheres fromendosome (E) to lysosomes (L) near nucleus (N);

FIG. 39 shows flow cytometry data, elucidating the effect of size andsurface modifications on the internalization efficiency of hollowspheres into HUVECs and HUASMCs cells. Flow cytometry data, elucidatingthe effect of size and surface modifications on the internalizationefficiency of spheres into (A) HUVECs and (B) HUASMCs at 12 hoursincubation;

FIG. 40 shows high content analysis showing internalization ofPEGylated, neutral and negatively charged hollow spheres with HUVECsover time. High content analysis showing internalization of PEGylated,neutral and negatively charged spheres with HUVECs over a time courseperiod of 6, 12, 24 and 48 hours. Data is represented as the mean +/−standard deviation (n=3, p<0.05);

FIG. 41 shows high content analysis showing internalization ofPEGylated, neutral and negatively charged hollow spheres with HUASMCsover time. High content analysis showing internalization of PEGylated,neutral and negatively charged spheres with HUASMCs over a time courseperiod of 6, 12, 24 and 48 hours. Data is represented as the mean +/−standard deviation (n=3, p<0.05);

FIG. 42 shows the effect of hollow sphere size and surface charge on %haemolysis after incubation with human erythrocytes. % Haemolysis afterincubation with human erythrocytes with (A) effect of size and (B)effect of surface charge. Data is represented as the mean +/− standarddeviation (n=4). * indicates a statistically significant differentbetween samples with p<0.05;

FIG. 43 shows the effect of hollow sphere size and surface charge onplatelet activation as indicated by sP-Selectin release. Plateletactivation as indicated by sP-Selectin release (A) effect of size and(B) effect of surface charge. Data is represented as the mean +/−standard deviation (n=4). * indicates a statistically significantdifference between samples with p<0.05;

FIG. 44 shows the effect of hollow sphere size and charge on complementactivation as indicated by C3a release. Complement activation asindicated by C3a release (A) effect of size and (B) effect of surfacecharge. Data is represented as the mean +/− standard deviation (n=4). *indicates a statistically significant difference between samples withp<0.05;

FIG. 45 shows the effect of hollow sphere size and charge on plasmarecalcification time, quantified using calculation of the point at whichthe recalcification profile reaches half of the maximum absorbancevalue. Plasma recalcification time, quantified using calculation of thepoint at which the recalcification profile reaches half of the maximumabsorbance value with (A) showing effect of size (B) showing effect ofsurface charge. Data is represented as the mean +/− standard deviation(n=4). * indicates a statistically significant difference (p<0.05), **indicates a statistically significant difference (p<0.01).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides biodegradable mono-dispersed nanoshells andnanospheres and a process for the preparation of such nanoshells andnanospheres. In particular the nanospheres of the invention are suitablefor use as vehicles to carry biomolecules, therapeutic agents and/orimaging agents to specific sites in the body. In order that theinvention may be more readily understood, the following examples aregiven by way of illustration only.

Example

Materials and Methods

Styrene, poly(vinyl pyrrolidine) (Mw 36,000), absolute ethanol,methanol, azobis-(isobytyrontile) (AIBN), sulfuric acid (H₂SO₄), aceticacid (AcOH), tetrahydrofuran (THF), chloroform (CHCl₃), FITC, DMEM cellmedia, Hanks PBS, 3T3 fibroblast cell line, bovine serum albumin,penicillin and streptomycin, collagen I, polystyrene beads (Gentaur,PP-025-100), chitosan (low molecular weight, 90% of deacethylation),polyglutamic acid (PGA), plasmid DNA (pDNA), Gaussia Luciferase (GLuc,New England BioLabs) and PicoGreen® (Invitrogen, P11496).

Chitosan (1 L, 0.5% w/v in 1% AcOH) was purified by precipitation from asolution adjusted to pH 7 using NaOH (sodium hydroxide). Theprecipitated chitosan was filtered using a strainer, washed withdistilled water and freeze-dried overnight. A solution at the desiredconcentration (0.5% w/v) was prepared in 1% AcOH.

Polystyrene (PS) beads 100 and 300 nm, phosphate buffered saline (PBS),2-(N-morpholino)ethanesulfonic acid (MES), N-hydroxysuccinimide (NHS),trypsine EDTA, methylthiazolyldiphenyltetrazolium bromide (MTT),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and2-methoxyethylamine (MEA). PS beads 510 and 1000 nm (GENTAUR, Brussels).PA series polyethylene glycol (PEG) (3400 Da) (Sunbright, NOFcorporation, Japan), agar low viscosity resin kit (Agar Scientific Ltd,Essex, UK). K3E and 9NC vacutainers (BD, Dublin, Ireland), enzyme linkedimmunosorbent assay (ELISA) kit for human soluble P-selectin(sP-selectin), immunoassay (R&D Systems, Minneapolis, USA). Human C3aELISA kit BD OptEIA_ from BD Biosciences-Pharmanigen (San Jose, Calif.,USA), fluorescein isothiocyanate (FITC), TO-PRO-3 iodide and bovineserum albumin (BSA) from Invitrogen (Dublin, Ireland). Endothelial cellgrowth medium-2 (EGM-2) and smooth muscle cell growth media (SmGM-2)(Lonza, England, UK), DNase free water, agarose, sodium bicarbonate,phosphate buffered saline (PBS), poly-D-glutamic acid (PGA),biocinchoninic acid assay (BCA) kit, sodium dodecyl sulfate (SDS), 30%acrylamide-bis and ammonium persulfate (Sigma-Aldrich, Ireland).Tetramethylethylenediamine (BIO-RAD (USA)) and PS beads of 500 and 1000nm (GENTAUR, Brussels). Quant-iT™ PicoGreen® dsDNA kit, fluoresceinisothiocyanate (FITC) and SimplyBlue™ (Invitrogen, Ireland),trinitrobenzene sulfonic acid (TNBSA) (Pierce, USA). Gaussia luciferaseplasmid (GLP), green fluorescence protein (GFP) plasmid and gaussialuciferase assay kit (New England BioLabs (UK)) agar low viscosity resinkit (Agar Scientific (UK). Ca²⁺-independent microbial transglutaminase(mTGase) (Activa®WM (Japan)), poly (2-dimethyl-aminoethylmethacrylate)(PDMAEMA)-block-poly ethylene glycol methyl ether methacrylate(PEGMEMA)/ethylene dimethacrylate (EDGMA), Amine terminated 4-arm StarPEG (GenKem Technology USA). branched PEI, sodium sulphate,Epichlorohydrin and Tween 20 (Sigma Aldrich Ireland).

Purification of mTGase

Ca²⁺-independent mTGase was purified as previously described. Briefly,the enzyme sample was dissolved in 20 mM sodium acetate buffer pH 5.8 ata concentration of 500 mg/ml and added to a glass column (1.5×30 cm)containing CM52 cation exchange resin (Whatman, UK) pre-equilibratedwith the above buffer at a flow rate of 2 ml/min. The sample was washedwith 2 column volumes of the same buffer and eluted by a gradient of 10column volumes from 0 to 0.5 M NaCl. The samples were analyzed at 280 nmfor protein and the pooled fractions were concentrated, dialysed intoPBS and analyzed for enzyme activity using the transglutaminasecolorimetric microassay kit (Covalab, UK) and purified guinea pig TGase(control) with known units of activity as standard (where 1 unit willcatalyze the formation of 1 pmole of hydroxamate at pH 6.0 at 37° C.using L-glutamic acid γ-monohydroxamate as the standard).

Preparation of Monodisperse Polystyrene Nanoparticles

Styrene (20 ml) was purified by treating with 20 ml of 20% aqueoussolution of sodium hydroxide at 10° C. The upper styrene layer wasseparated and washed with water (20 ml×5) and dried under anhydroussodium sulphate. It was then dispersion polymerized in aqueous alcoholicmedium. The medium used for the dispersion polymer is a mixture of 75vol % absolute ethanol and 25 vol % water. Poly(vinyl pyrrolidine)(36000 Mw) (0.4-2 wt % on medium) was dissolved in the medium at roomtemperature. Styrene (10 wt % on medium) is added to the medium followedby azobis-(isobutyrontrile) (AIBN) (0.1-1.0 wt % on styrene). Thereaction mass was stirred at 120 RPM speed at room temperature for 1hour. Later the temperature of the reaction mixture is raised to 70° C.where it is maintained for 24 hours. The reaction mixture is dilutedwith double the amount of reaction mass with methanol and thencentrifuged at 4000 RPM and at 8° C. temperature.

Sulfonation of Polystyrene Nanoparticles

The surface of the polystyrene particles was functionalized bysulfonating the particles. Sulfonation was carried out by treating theparticles with sulphuric acid. As the polystyrene particles arehydrophobic and light-weight, they stay on the surface of the sulphuricacid medium. To get uniform sulfonation, the particles should beuniformly dispersed in the sulfonation medium. Hence, the particles (1.7g) are first treated with 60 ml sulphuric acid and sonicated to ensurehomogeneous dispersion. The particles at this time are dispersed in themedium. The temperature of the reaction mixture is raised to 40° C. Itis maintained for 18 hours under stirring. The reaction mass iscentrifuged many times at 6,000 to 7,000 RPM. The particles are washedwith ethanol and centrifuged to get the sulfonated polystyreneparticles.

Preparation of Nanoshells

The sulfonated nano-particles were dispersed in the medium (solvent) inwhich the natural polymer is soluble. In the present example, theparticles (500 mg) were dispersed in 10 ml of 1% acetic acid solution inwater. A solution of the desired natural polymer was prepared in 1%acetic acid solution at 0° C. 10 ml of the dispersed solution of theparticles was then treated with 10 ml of the polymeric solution andagitated on a mechanical shaker for 24 hours at 0° C. Agitation ensuresa good coating is obtained on the surface of the template, i.e. thesulfonated polystyrene bead. The mass was then centrifuged and the solidwas washed with 1% acetic acid aqueous solution to remove the unreactedpolymeric solution. The solid particles were separated and dispersed in1% acetic acid solution 10 ml.

These particles can be either cross-linked at this step or later.Several coating steps can be carried out in a layer-by-layer process toobtain multi-layer particles. Crosslinking can occur before, after orduring layering. The therapeutic can also be added before cross-linking.

To crosslink, the particles were treated with a 1% vol solution of Amineterminated 4-arm Star PEG in ethanol:water (80:20 by volume) at roomtemperature for 5 hours. A ratio of 1:1 cross-linker to polymer wasused.

In a separate round-bottom flask, 77 mg (0.52 mmol, 1.7 eq) ofpolyglutamic acid were dissolved in 20 mL of2-(N-morpholino)ethanesulfonic acid solution (MES, 0.05M, pH 5.5),followed by the addition of 26 mg (0.24 mmol, 0.8 eq) ofN-Hydroxysuccinimide (NHS) and 40 μL (0.24 mmol, 0.8 eq) ofN-Ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC). The mixture wasstirred for 5 minutes at room temperature, added to the suspension ofchitosan-coated beads and agitated for 24 hours in a mechanical shaker.

To prepare the shell, the polystyrene core was removed by treating theparticles in 10 ml of 1% acetic acid solution with 20 ml THF:water(80:20 by volume) for 24 hours at room temperature. The particles werewashed with a 1% vol solution of acetic acid in a THF:water (60:40 byvolume) solution several times. The mass was centrifuged and the shellswere collected.

Alternatively, to prepare the shell, the polystyrene core was removed bytreating the particles with THF. Briefly, the cross-linking mixture wasdiluted by a factor of three with THF and centrifuged four times at 3000G. The collected pellets were then washed 3-4 times with THF,centrifuged and dried under vacuum.

Fabrication of Hollow Spheres

Hollow spheres were fabricated using a template based method. Briefly,the fabrication method includes three processes: coating (describedabove), cross-linking and dissolution of the core to obtain the hollowsphere. The sulfonated beads were dispersed in the medium (solvent) inwhich the natural polymer is soluble. In the present example, the beads(500mg) were dispersed in 10 ml of 1% acetic acid solution in water. Asolution of the desired natural polymer was prepared in 1% acetic acidsolution at 0° C. Coated beads were then cross-linked using microbialtransglutaminase (mTGase). Finally, PS beads were dissolved using THF toobtain the hollow spheres.

Hollow spheres were fabricated as follows: Briefly, a 0.5 wt % solutionof polymer in 1% (v/v) acetic acid was added to a colloidal solution ofsulfonated PS beads of various sizes (100, 300, 500 and 1000 nm) and themixture was then shaken for 24 hours at 4° C. PGA (1.7 equiv) in MES(0.05 m, pH 5.5) was mixed for 5 min with NHS (0.8 equiv) and EDC (0.8equiv). This was then added to the polymer solution and the solution wasstirred for 24 hours. Cross-linking reaction occurred over 24 hours. Toobtain a surface negative charge on the native hollow spheres anadditional 0.7 equiv PGA was added to the polymer solution during thefabrication process. Finally, to obtain hollow spheres, PS cores weredissolved with a 1% vol solution of acetic acid in a THF:water (60:40 byvolume) solution and dried under vacuum to evaporate excessive solvent.The prepared nanospheres were cross-linked with various cross-linkersusing EDC/NHS, 4 arm Star PEG and epichlorohydrin and optimized. Severalcoating steps can be carried out in a layer-by-layer process to obtainmulti-layer spheres. Cross-linking can occur before, after or duringlayering.

Cross Linking Natural Polymer with Pre-Activated Dendrimer

The solution of natural polymer was treated with a solution of 2 mg/mlof activated functional dendrimer in DMF for 12-24 hours at 4-5 C. Thecross-linked scaffold was then freeze dried and washed with 50:50triethylamine:water or dilute ammonium hydroxide at 4-5 C for 12 hoursand then water for 12 hours, finally with neutral buffer.

Dendrimeric System Development

Fluorescein (130 ml, 3 mg/ml in acetone) was added to PAMAM generation 1(130 ml, 10.9 mg/ml in water) and mixed for 24 hours in the dark at roomtemperature. The reaction was monitored by thin layer chromatography.Then, the sample was extensively dialysed for 4 days, lyophilized andstored at −20° C. until use.

Surface Modification of Nanoshells

The surface functional groups of the polymer nanoshells were modifiedwith labelling agent fluorescein isothiocyanate FITC by dispersing thenanoshells in a solution of FITC in 10 mM Tris-HCL for 24 hours at roomtemperature. The shells were centrifuged at 5000 rpm and 8° C. andwashed 5 times with 10 mM Tris HCL solution for 10 min. The shells werethen sterilized and stored.

Zeta Sizer Analysis

Zeta sizer (Malvern, Nano-ZS90) was used to characterize the polymericcoating over sulfonated PS beads. 500 nm beads were sulfonated and usedfor the coating experiment. of different amounts to that of a fixedquantity of PS beads was used. The ratios used were 50:1, 75:1 and 100:1of PS to beads (μg/mg).

Alteration of Surface Charge and Function of Hollow Spheres

Spheres of all the four sizes were used for surface modifications. Forneutralization, native spheres were covalently cross-linked with MEA.Briefly, 50 mg of polymer/PGA hollow spheres (0.086 mmol of carboxylicgroup) were dispersed in MES buffer (2-3 ml, pH 5.5) in a round bottomflask and 22.24 μl of MEA (0.258 mmol of amino group), EDC (0.172 mmol)and NHS (0.172 mmol) were then added. The mixture was stirred overnightat room temperature and dialyzed to remove the unreacted chemicals.Surface PEGylation of these hollow spheres was performed usingpropylamine-functionalized amino-terminated PEG. 0.043 mmol of PEG wasmixed with 0.086 mmol hollow spheres with EDC (0.086 mmol) and NHS(0.086 mmol) in MES buffer (pH 5.5). The mixture was then stirredovernight and dialyzed to remove the chemicals that did not react.Surface charge was analyzed in mV using zeta sizer (NanoZS, Malvern)after surface modifications of all the spheres. Briefly, polymer wasdissolved in 2% Tween 20 in PBS in the concentration of 10 mg/ml and 20%w/v sodium sulphate was added drop wise in slight excess until itconvert from a clear solution to turbid solution. The preparednanospheres were hardened by further crosslinking with variouscrosslinkers including EDC/NHS-4 arm Star PEG and epichlorohydrin andoptimized.

Fabrication of Different Sizes of Hollow Spheres and FITC Labelling

Spheres were observed under transmission electron microscopy (TEM) foranalyzing their internal structure and size. FITC labeling was performedas described as follows: PGA was labeled with FITC prior tocross-linking step during the hollow sphere fabrication process.Briefly, a weight ratio of 1:40 of FITC to PGA was kept shaking at 4° C.for overnight. The unbound FITC molecules were removed by dialyzing.FITC labelled PGA was then used to fabricate the hollow spheres.FITC-dextran loaded nanospheres were also prepared following the sameprotocol.

Encapsulation of Therapeutics

The nanoshells were sterilized and dispersed in a solution of specifictherapeutic agent in a small volume of 10 mL Tris-HCL and 1 mM EDTAbuffer solution for 1-7 days. The therapeutic agent was entrapped insidethe cavity of the shells by physical diffusion and functionalinteractions. The shells were centrifuged and separated. The nanoshellswere finally washed with a mixture of 10 mM Tris-HCL and 1 mM EDTAbuffer. The entrapment efficiency of the therapeutic agent was measuredby dissolving the uncrosslinked nanoshells and measuring the releasedplasmid DNA against UV absorbance.

The molecule of interest may, alternatively, be encapsulated by emulsiontechnique. Briefly, nanoshells are solubilised in CHCI₃, pDNA and fewmillilitre of PVA solution (9%) are added and emulsified by sonicationduring 15 seconds and stirring for 3 hours to evaporate CHCI₃. Theformulation mixture is then centrifuged and complexes are washed threetimes with ultra pure water.

Example of DNA Encapsulation

DNA encapsulation was accomplished using one of the two followingprotocols outlined below:

Protocol 1: An 8 mg sample of nanospheres was suspended in 1 mL ofultra-pure water. A 1-2 mL volume of THF or CHCl₃ and 40 μg pDNA wereadded, and the mixture was agitated for 3-4 hours at room temperature.The resulting sphere/pDNA complexes were centrifuged (13,000 G) andwashed three times with absolute ethanol and twice with ultra-purewater.

Protocol 2: An 8 mg sample of nanospheres was suspended in 1 mL ofultra-pure water. A 1-2 mL volume of THF or CHCl₃ was added, and thesuspension was centrifuged (13,000 G). The particles were washed threetimes with absolute ethanol and twice with ultra-pure water, followed bythe addition of 40 μg of pDNA. The mixture was incubated over night at4° C. and then washed three times with ultra-pure water. The sphere/pDNAcomplexes were diluted to the desired concentration using DMEM media.

To increase pDNA loading efficiency of hollow spheres and provideprotection against endosomal degradation, polyplexes were prepared,mixing 10:1 weight ratio of a hyper branched block copolymer of poly(2-dimethyl-aminoethylmethacrylate) (PDMAEMA)-block-poly ethylene glycolmethyl ether methacrylate (PEGMEMA)/ethylene dimethacrylate (EDGMA)polymer and GLuc plasmid DNA in phosphate buffer solution pH 7.4.

Electrophoresis Gel

A 0.9% agarose gel (stained with SYBR safe) was prepared with TEA (1×)buffer and the migration was carried out under a voltage of 100 V.

Encapsulation Ratio

Following DNA encapsulation, the amount of pDNA remaining in the washsolutions was quantified using the PicoGreen® assay, which was performedaccording to the manufacturer's instructions.

pDNA Integrity

A 1 mg sample of sphere/pDNA complex (obtained using protocol 1) wassuspended in reaction buffer (Sigma DNase1 kit). A 1 μL solution ofDNase1 was added, and the mixture was incubated for 15 min. The reactionwas halted by the addition of 1 μL of stop solution, and the mixture washeated to 70° C. for 10min. The integrity of the pDNA was verified usinggel electrophoresis (0.7% agarose).

pDNA Release

A 1 mg sample of sphere/pDNA complex (obtained using protocol 1) wassuspended in 10% FBS complemented DMEM and incubated under shaking at37° C. At every time point, the suspension was centrifuged and a sampleof the supernatant was taken. The amount of pDNA remaining in the samplesolutions was quantified using the PicoGreen® assay (performed accordingto the manufacturer's instructions), and the percentage of release wasdetermined by the quantity of encapsulated pDNA.

Enzymatic pDNA Release

The enzymatic release study was performed by incubating 1 mg ofsphere/pDNA complex with protease (20 units per mg of complex) in 1.5 mlof buffer (10 mM sodium acetate buffer with 5 mM calcium acetate, pH7.5) at 37° C. At every time point, the mixture was centrifuged (16,000G) and 100 μl of sample was taken. The total volume was kept at 1.5 mlby adding 100 μl of fresh buffer. The amount of pDNA was quantifiedusing the PicoGreen® assay.

Uptake of Nanoshells by Cells

Nanoshell uptake into cultured 3T3 cells was examined by tracing FITCfluorescence labeled nanoshells at various concentrations using confocallaser scanning microscopy (CLSM) and flow cytometry (FACS).

For CLSM, the cells were seeded on Lab-Tek chamered coverglasses andincubated with fluorescence-labelled chitosan and collagen nanoshells ata final concentration of μg/ml for 6 hours at 37° C. and 4° C.respectively. After washing the cells with PBS, the cell membrane wascontrasted with 0.0005% (m/v) solution of tetramethyrhodamine(TRITC)-labelled lectin (concanavalin A-tetremethylrhodamine conjugate).The cells were fixed with a 4% solution of paraformaldehyde for 10 minand covered with 10% mowiol 488 (Clariant), 2.5% 1,4-diazabicyclo[2,2,2] octane and 25% glycerol in 0.2M Tris buffer. Confocal microscopywas performed with a Letiz microscope and a TCS True Confocal Scannerequipped with a krypton-argon laser. This can show if DNA has beenencapsulated into the shell and if complexes are entering into the cell.The technique can also be used to determine the route that complexes areusing and where in the cell the DNA is released.

For FACS, cultivated cells were incubated for 6 hrs with FITCfluorescence-labelled nanoshells in culture at 37° and 4° C. and werethen trypsinised. Alternatively, the cells were trypsinised firstfollowed by incubation at various nanoparticle concentrations insuspension at 37° and 4° C. for 6 hours. Nanoparticle concentrations of5, 10 and 50 μg/ml were used. The cell membrane was stained with aTRITC-concanavalin A conjugate. The cells were analysed with a FACScalibur flowcytometer. Significance was calculated from raw data withthe Wilcoxon-Mann-Whitney test. Internalisation of the nanoparticles wasseen.

The morphology of the samples was studied using scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM).

Cell Study

3T3 fibroblast cells were cultured in DMEM supplemented with 10% fetalbovine serum (FBS) and 1% penicillin-streptomycin and maintained in ahumidified atmosphere containing 5% CO₂ and 95% air at 37° C. Cells werethen seeded, 24 hours prior to the experiment, in a 96-well plate(10,000 per well) and incubated with the dendrimer-fluorescein sample(30 μmol per well) for 3 hours. Cells were washed (with PBS buffer),fixed with 4% paraformaldehyde/2% sucrose and then stained withrhodamine phalloidin. Confocal microscopy was used for this analysis.

Mass Spectral Characterisation

Sample characterisation was carried out using mass spectrometry. Massspectral analysis involves the formation of gaseous ions from an analyte(M) and subsequent measurement of mass-to-charge ratio (M⁺) of theseions. Mass spectrometry plays an increasingly important role in polymeranalysis, because of its high sensitivity, broad dynamic range,specificity, and selectivity.

Scanning Electron Microscopy (SEM)

Scanning Electron Microscopy—Energy Dispersive X-ray Detection (SEM-EDX)images were obtained using a Hitachi S-4700 field emission microscopeoperating with a beam voltage of 15 kV and equipped with a backscatteredelectron detector. A drop of particles was placed on adhesive carbontabs mounted on SEM specimen stubs and dried. The specimen stubs werethen coated with 5 nm of gold by ion beam evaporation using an EmitechK550 coating system.

Transmission electron microscopy (TEM)

TEM measurements were performed using a Hitachi H-7500 microscope. TheTEM samples were prepared by depositing a diluted particle suspension ona carbon-coated copper grid, followed by air-drying.

Hollow spheres of the four different sizes were observed undertransmission electron microscopy (TEM) (Hitachi H7500) and scanningelectron microscopy (SEM) (Hitachi S-4700). Additionally, ultrastructureof solid spheres of was observed under TEM. Further visualization ofinternalization was obtained by TEM. Cells were incubated with 50 μg ofspheres for 24 hours. The TEM images showed transparent and squeezedhollow spheres (FIG. 26). The sizes of the hollow spheres as estimatedfrom their TEM images are 100±13, 300±26, 500±34, 1000±90 nm. Thesurface charge of all the sizes of hollow spheres as measured using zetasizer were in the range of −20 to −27 mV (FIG. 26). This demonstratesthat these hollow spheres can be used as a potential reservoir systemfor various therapeutic molecules.

Fourier Transform Infrared (FTIR) Analyses

The infrared spectra were obtained using a Shimadzu FTIR-8200 FourierTransform Infrared Spectrophotometer. The samples were analysed withoutany preparation.

Blood Compatibility

A major challenge for the systemic delivery of synthetic vehicles forgene delivery is their lack of stability in the blood stream, theirdegradation, and clearance by the reticuloendothelial system, whichmakes the elucidation of their interaction with blood componentsessential. Several interactions with the family of spheres wereinvestigated to determine the potential systemic delivery in vivo of thehollow spheres.

Characterisation of Sulfonated Polystyrene Beads

Each step of the hollow-sphere production process was characterisedusing one or more techniques such as SEM, TEM and FTIR. FIG. 10 is anelectron micrograph depicting the structural morphology of the beads.The particles are consistent in shape and size with a mean diameter of300 nm. The sulfonated beads were evaluated using FTIR. FIG. 11 containsinfrared spectra of the polystyrene beads before and after sulfonation.The appearance of characteristic peaks at 3300 cm⁻¹ and 1200 cm⁻¹indicates that the beads were significantly sulfonated.

Study of Effect of Ratios of Cross-Linker to Polymer

To illustrate the property differences induce by the particularcrosslinker used, the effect of the ratio of cross-linker to polymerused for cross-linking the polymeric nanoshells on the physicalproperties of the nanoshells was investigated. Table 1 provides asummary of the cross-linker to polymer ratios investigated.

TABLE 1 A summary of the cross-linker to polymer ratios investigated NH₂group COOH group EDC/NHS Batch n^(o) (equivalent) (equivalent)(equivalent) 1 1 4 0.8 2 1 3 0.8 3 1 2 0.8

As discussed above, the sulfonated beads were coated with chitosansolution. At low concentrations, SEM micrographs revealed discretechitosan-coated beads (FIG. 12A), but when the concentration wasincreased, the polymer covered the beads. Because of this highconcentration of chitosan, the structure appears to look like aggregatedbeads resulting, after cross-linking, in a scaffold-like structure (FIG.12C, 12D), which is undesirable in the present objective.

After coating, the polymeric layer was cross-linked using EDC/NHS andthe sulfonated beads were removed to obtain hollow spheres. The presenceof the hollow core was confirmed by the disappearance of thecharacteristic peaks of polystyrene beads (FIG. 13) and by the flatshape of the sphere (FIG. 3A, 3B, 3C) due to the high vacuum in the SEMchamber. The results show that when the cross-linker to polymer ratio isincreased (equivalent quantity of COOH group decreases) the rigidity ofthe membrane increases. In fact, nanoshells obtained with 1/4 (NH₂/COOH)ratio are totally crushed (FIGS. 3A, 4A). Nanoshells with 1/3 ratio areless crushed and look like “deflated balls” (FIGS. 3B, 4B). From theseresults it is concluded that the optimum ratio of cross-linker topolymer to produce well-defined nanoshells is 1/2 (NH₂/COOH) (FIGS. 3C,4C) as shown by the electron microscopy images in FIG. 3.

TNBSA Assay and SDS-PAGE for Cross-Linking

The cross-linking of hollow spheres with mTGase was illustrated usingTNBSA, a hydrophilic modifying reagent for the detection of primaryamines in samples containing amino acids, peptides or proteins. TNBSAassay was performed to characterize the mTGase cross-linking of hollowspheres and also to quantify the amount of free amino groups availableon the surface. The data indicate that mTGase cross-linking leaves ahigher proportion of free amino groups than that of glutaraldehyde (FIG.27).

Effect of Degree of Crosslinking on Physical Properties of theNanosphere

The nanospheres according to the invention illustrate that cross-linkingof the polymers forming the nanosphere improves the stability andmechanical integrity of the nanosphere. The degree of crosslinking canaffect various physical properties of the nanosphere such as thepermeability of the sphere wall, the rate of degradation of the sphere,and the rate of release of agents encapsulated within the sphere forexample. Thus, FIG. 5 shows the DNA encapsulation ratio comparisonbetween chitosan nanospheres crosslinked with glutaraldehyde andchitosan/PGA nanospheres. Different cross-linkers and ratios of crosslinker display difference release rates.

Dendrimeric System Development

The fluorescein-dendrimer reaction was monitored using thin layerchromatography. The TLC plate is shown in FIG. 6. The objective was tolink one FITC probe onto the dendrimer in order to be able to track itwithout changing its properties. Due to the excess of free amine groups(7 per molecule), the conjugate (samples 1, 2 & 3) barely moved from theorigin. The unbound FITC (F) probe migrates near to the solvent front.

Mass spectrometry was used to characterise the conjugate. The massspectra (FIG. 7) revealed the presence of a peak at 908.5. The 0.5difference in mass to charge ratio (m/z) between the neighbouring peaks(isotopic difference) indicates that the sample is ‘doubly charged’(z=2). The visible molecular weight of the complex is 908.5 doubled plustwo protons or 1819 (908.5*2+2). This value matches the theoreticalmolecular weight of 1819.25 (the sum of the molecular weight's of PAMAMG1 and FITC) confirming that the fluorescein-dendrimer conjugate wasformed correctly.

Confocal microscope studies were carried out without (FIG. 8) and with(FIG. 9) FITC labelled PAMAM. Due to their positive charge ratios (FIG.9) an efficient uptake of the complexes into the cells was seen. Thisresult was confirmed by the absence of green fluorescence (FITC) as seenin the control micrographs (FIG. 8).

Zeta Size Analysis

Zeta potential analysis of 1000 nm hollow spheres after loading showedan increase in surface charge from negative to a value near to +7 mV ,indicating the presence of polyplexes on their outer surface (FIG. 28).The amount of pDNA on the outer surface was found to be 8-10% of thetotal amount loaded as assayed by Picogreen® (FIG. 29). A similar trendwas found in all other spheres. The polyplex loaded hollow spheres werethen analyzed under TEM, where they appeared to be more compact and darkthan that of unloaded hollow spheres (FIG. 30E).

Zeta sizer (Malvern, Nano-ZS90) was used to characterize the polymericcoating over sulfonated PS beads. 500 nm beads were sulfonated and usedfor the coating experiment. of different amounts to that of a fixedquantity of PS beads was used. The ratios used were 50:1, 75:1and 100:1of PS to beads (μg/mg). Size and zeta potential of the coated and PSbeads were analyzed to prove the coating. In addition, surface charge ofhollow spheres was determined. The surface charge of all the sizes ofhollow spheres as measured using zeta sizer were in the range of −20 to−27 mV (FIG. 26).

Encapsulation of Therapeutics

pDNA Encapsulation Efficiency and Release

Two encapsulation protocols were attempted. The second method describedabove (protocol 2) involved the addition of the pDNA at the end of theprocess in an attempt to encapsulate the pDNA by diffusion. This methodwas unsuccessful due to the low permeability of the membrane. However,when the pDNA was added during the emulsion step according to theinitial process (protocol 1), the encapsulation was more efficient. Theresults showed that the polymeric shell was capable of encapsulatingapproximately 92% of the pDNA (FIG. 14). The polymeric shell pDNAcomplex was also confirmed by confocal microscopy which showedcollocation of FITC-labelled polymeric shell (indicated by green areason the micrograph) and ethidium bromide labelled pDNA (indicated by redareas on the micrograph) as shown in FIG. 14 B. The fluorescencemicroscopy along with FACS study demonstrated the uptake behavior ofHUVEC and HVSMC. The uptake of spheres was seen within 6-12 hrs ofincubation period (FIG. 15). A yellow color (mix of green and red)represented the colocalisation of spheres inside the cell. Thebrightness of this yellow color showed the level of internalization.

In vitro studies demonstrated the spheres significant capacity toencapsulate pDNA with efficiency of up to 95% (FIG. 16A). The potentialof these shells for gene transfection was investigated by studyingcellular uptake. It was observed that for the uptake by cells, thespheres need a longer incubation time (6-12 hours) than cationicpolyplexes (FIG. 16B). This is because of their negative/neutral surfacecharge ratios, which allow them to stay in presence of serum withoutaggregation or precipitation. Following encapsulation, the integrity ofthe pDNA was checked using gel electrophoresis (FIG. 17). After washing,no free pDNA was left in solution and no degradation band appeared onthe gel. The degree of protection afforded by the complexes and theirability to release the pDNA were also examined. In order to mimic the invivo conditions (presence of proteins), the release was performed inFBS-containing medium. No release of pDNA was observed, even after threedays of incubation (FIG. 18). The integrity of the complexes was checkedby SEM, and TEM and no degradation or modification of the shape wasobserved. On the other hand, in presence of enzyme, Picogreen® assayshowed a release of 30% of the encapsulated pDNA after 72 hours withoutany damage FIG. 19. To quantify the polyplex, it was treated with PGA of10 mg/ml concentration. Results from agarose gel electrophoresis wereconsistent with that of PicoGreen® where a similar band pattern wasobserved between pDNA and PGA treated polyplex. (FIG. 31). Tocharacterize loading, 1000 nm hollow spheres were incubated with aninitial 20 μg of pDNA alone and polyplex containing the similar amountof pDNA. The loading efficacy of the NUI Galway hollow nanospheretechnology was compared to that of solid spheres of similar size. Solidspheres of around 300 nm were fabricated by incubating 1 mg/ml ofpolymeric solution with 20 μg of pDNA at 37° C. The solid spheres werestable after cross-linking with mTGase and adding 20% of THF (FIG. 32).pDNA of the same amount was incubated with 300 nm hollow spheres for 12hours. The loading efficiency was analyzed by quantification of pDNA inthe supernatant using PicoGreen® assay (FIG. 30A). Irrespective of anegative surface charge, the hollow spheres showed around 25% higherpDNA loading than the solid spheres. In the case of polyplex, free pDNAwas quantified for the loading efficiency. The pDNA was released fromthe polyplex by treating with 10 wt % of polyglutamic acid (PGA) (FIG.33). The method was used to allow accurate quantification of pDNAloading. The result showed that almost 98% of pDNA was loaded within thehollow spheres in the form of polyplex as compared to 54% of pDNA alone(FIG. 30 B). The maximum loading was seen in the case of 1:80 which didnot show any significant difference when increased to 1:160. Thus theresult showed an approximate 69 μg of pDNA/mg of hollow sphere as themaximum loading efficiency. In addition, the loading was found to besimilar in all the four sizes of hollow spheres tested (FIG. 30 D).

Release studies were performed in the presence of enzymes at 37° C. Therelease pattern was observed up to 192 hours for all sphere sizes.Spheres of 1 mg dry weight containing 60-70 μg of pDNA were used forthis study. No significant difference in the release pattern wasobserved for differences in sphere sizes up to 96 hours. And a total of6-7 μg of pDNA was released. However, the release profile was found tobe different at 192 hours, where the 100 nm and 300 nm hollow spheresshowed less release than 500 and 1000 nm hollow spheres (FIG. 34 A). Thehollow spheres were then treated with appropriate enzymes which areabundantly found inside the human body in diseased conditions. Theenzymatic treatment released polyplexes much faster than untreatedspheres (FIGS. 134 B and E). The control hollow spheres without anytreatment of enzymes had a release of 20% of pDNA at 72 hours, whereasthe release for enzyme treated spheres were found to be 85, 71% and 70%respectively. Degradation of the hollow spheres was observed under SEM(FIG. 34 C2).

DNA Protection

The ability of the spheres to protect the pDNA was studied using DNase1enzyme. After 15 minutes of incubation with DNase1, no apparentdegradation of pDNA was seen using gel electrophoresis (FIG. 20).Moreover, no free or degraded DNA was observed after complex extraction(FIG. 20), implying that no pDNA was extracted or released from theshell.

Cell Uptake

The kinetic uptake of FITC labelled spheres (100 to 600 nm) by HumanUmbilical Vein Endothelial Cells (HUVEC) and Human Vascular SmoothMuscle Cells (HVSMC) was studied. Confocal micrographs shows that bothcell lines which were stained in red) are uptaking FITC labelled Spheres(which were stained in green) from 6 to 12 hours (FIG. 21). Moreoverwhen the incubation time is increased, results show an increase inuptake. These results have been confirmed by flow cytometry which showsa significant increase in the fluorescence.

Blood Compatibility

A major challenge for the systemic delivery of synthetic vehicles forgene delivery is their lack of stability in the blood stream, theirdegradation, and clearance by the reticuloendothelial system, whichmakes the elucidation of their interaction with blood componentsessential. Several interactions with the family of spheres wereinvestigated to determine the potential systemic delivery in vivo of thehollow spheres. The nanoshells according to the invention were testedfor biocompatibility. The results are shown in FIGS. 22 to 25. Theeffect of various parameters such as the charge on the spheres and sizeof the spheres, on haemolysis and platelet activation was studied.

All the experiments for haemocompatibility were performed using humanvenous blood from healthy volunteer donors. Haemolysis was evaluated onvenous blood anticoagulated with EDTA. Within 2 h, the erythrocytes werewashed and resuspended in PBS at a ratio of 1:10. Functionalised sphereswere added in triplicate to the erythrocyte suspension to a finalconcentration of 50 μg/ml. TritonX was tested as the positive control.After incubation at 37° C. for 2 h, the samples were centrifuged at 1000rpm for 15 min to remove the non-lysed erythrocytes. The supernatantswere collected and analyzed for the released haemoglobin byspectrophotometric determination at 540 nm. To obtain 0 and 100%haemolysis, the erythrocyte suspension was added to PBS and to TritonX,respectively. The degree of haemolysis was determined by the followingequation:haemolysis (%)=(Abs−Abs₀)/(Abs₁₀₀−Abs₀)×100,where Abs, Abs₀ and Abs₁₀₀ are the absorbance of the test samples, asolution of 0% haemolysis and a solution of 100% haemolysis,respectively.

Supernatants obtained by centrifugation at 85 G (10 min) followed by oneat 140 G (10 min) were reunited to compose the Platelet Rich Plasma(PRP). The number of platelets was determined under microscope with ahaemocytometer after 1/100 PRP dilution with ammonium oxalate.Functionalised spheres were added to PRP to a final concentration of 100μg/ml. PBS was tested as the negative control. The samples were rotatedfor 1 H at 37° C., then immediately centrifuged and the supernatant wasanalysed by Elisa test (#KHS2021, invitrogen) according to themanufacturer protocol.

The spheres according to the present invention are very suitable for invivo use due to their haemocompatibility. In fact, haemolysis graphs(FIGS. 22 and 23) demonstrate that chitosan/PGA spheres don't lysehaemoglobin (<1%). Moreover, this rate decreases for the smallest sizesand when the surface of the sphere is functionalised. Furthermore, theelisa detection experiment shows that the platelet activation decreasesafter functionalisation.

Platelet Activation

Platelet activation was measured by the concentration of sP-selectinlevels in the plasma and was determined using ELISA kit according to themanufacturer's protocol. Platelet-poor plasma (PPP) and Platelet-richPlasma (PRP) were used as control.

Complement System

To assess complement activation, the cleavage of complement component C3was monitored by measuring the formation of its activation peptide; C3adesArg, using a commercial C3a enzyme immunoassay kit.

Plasma Clotting Time

0.1 ml of the PPP and 40 μg of samples suspended in PBS were incubatedat 37° C. for 5 min in a 96 well plate. 0.1 ml of 0.025 m CaCl₂ solutionwas then added and the plasma solution was monitored for clotting bymanually dipping a stainless-steel wire hook coated with silicone intothe solution, to detect fibrin threads. Clotting times were recorded asthe time at which first fibrin strand formed on the hook. Plasmarecalcification profiles are used to mimic the intrinsic coagulationsystem in vitro. PBS was used as a negative control in this study.

Cell Viability Study

Spheres of all four different sizes were loaded with gaussia luciferaseplasmid (GLP). The GLP/polyplex loaded spheres of all the sizes showed asimilar cell viability to the control (untreated cell) in both ADSCs andhuman umbilical vein endothelial cells (HUVECs) after 48 hours, whereasthe cells treated with polyplex alone showed less cell viability asevident from the PicoGreen® assay (FIGS. 35 A and B). Polyplex loadedhollow spheres of all the sizes showed better expression than pDNA andpolyplex alone in both ADSCs and HUVECs (FIG. 35 B). In addition,similar luciferase expression was found for all the sizes without anysignificant difference in either ADSCs or HUVECs. FIG. 35 C showed analmost 10 times higher transfection level of polyplex loaded spheresthan the pDNA demonstrating the endosomal protection ability of polyplexloaded hollow spheres in ADSCs. The cellular internalization pathway ofthe polyplex loaded sphere was tracked inside ADSCs to assess themechanism of endosomal protection. ADSCs treated with 500 nm sphereswere fixed and embedded in resin after 6, 12 and 24 hour time points.The negatively charged spheres point was observed attaching on the cellmembrane (CM) after 6 hour time (FIG. 36 A) and were gradually engulfedby the CM (FIG. 36 B). FIG. 36 C shows the hollow spheres in an earlyendosome after 12 hours. The spheres were then observed in late endosomeor lysosome adjacent to the nucleus (FIG. 36 D). After 24 hours, thelysosome starts to degrade as a consequence of its internalized sphere,and the sphere also loses its shape and starts to degrade (FIG. 36 E).The hollow sphere eventually exits the degrading lysosome (FIG. 36 F).The hollow spheres can be used as a gene delivery depot system forspatial and temporal controlled pDNA release leading to a sustained,local delivery of therapeutic factors and also as a transfection agent.The polyplex used can protect pDNA from degradation, promote interactionwith cell membranes, and facilitate endosomal release via the protonsponge effect. Moreover, significantly higher amounts of gene can beloaded in hollow spheres. In addition, hollow spheres can be used forthe loading of both hydrophobic and hydrophilic drug molecules as theloading will be through a diffusion related process. The free —NH₂ and—COOH groups can be readily conjugated to targeting moieties forspecific applications.

Cellular Internalization Behaviour of Spheres

Characterization by Confocal Imaging

HUVECs and HUASMCs cells were incubated with FITC labelled hollowspheres. Confocal micrographs show co-localization of the negativelycharged FITC labeled spheres (green) within HUVECs and HUASMCs after 24hour incubation (FIG. 37). 100 nm and 300 nm spheres can be seen in theperinuclear region of the cells. The cell uptake of hollow spheres wasinvestigated by TEM. During time depended cellular tracking of thefluorescent nanospheres, fluorescence appeared to be distributedthroughout the cytoplasm inside the cells post 4 hours incubation withnanospheres in the presence of serum containing media (FIG. 37(D-G)).Cells incubated with 100 nm neutral hollow spheres for 24 hours wereobserved under TEM (FIG. 38). TEM micrographs show hollow spheres withinlysosomes of both the cell types (FIGS. 38 A and B). FIG. 38 Cillustrates the endocytic pathway of hollow spheres from early endosometo lysosome inside HUVEC.

Quantification by Flow Cytometry

Cells were then analyzed using flow cytometry for internalizationefficiency. The impact of size and surface charge on cellularinternalization was quantified at 12 hours following incubation usingflow cytometry. FIG. 39 shows internalization efficiency of sphereswithin HUVECs (FIG. 39 A) and HUASMCs (FIG. 39 B). 100 nm neutralspheres showed increased internalization in both cell types with 76% inHUVECs and 56% in HUASMCs. HUASMCs had reduced sphere uptake than HUVECsin all the sizes and surface modifications investigated. 300 and 500 nmspheres show similar internalization efficiency in both HUVECs andHUASMCs. 1000 nm spheres, regardless of surface charge had lowinternalization with 9-13% internalization in both cell typesinvestigated. Among all the sizes, negatively charged spheres presentedthe lowest uptake profile.

Quantification by High Content Analysis

HUVECs and HUASMCs were seeded on 96-well plates for high contentanalysis (HCA). FITC-hollow spheres were seeded and incubated fordifferent time points 6, 12, 24 and 48 hours. After the desiredincubation times cells were fixed and stained for nucleus using TO-PRO-3iodide. Finally, the sample wells were quantified using In Cell Analyzer1000 GE Healthcare for 420 nm (FITC) and 620 nm (TO-PRO-3 iodide). HCAenabled quantitative estimation of the internalization of FITC labellednanospheres of different parameters, including size, surface charges andtime points within HUVECs and HUASMCs. Cellular internalization wasestimated in terms of relative fluorescence. The results found that 100nm neutral spheres were significantly more internalized (p<0.05) whencompared with other sizes, for PEGylated and neutrally charged spheresin both cell types, which is consistent with flow cytometric data, andshowed a constant increase of internalization over time from a relativefluorescence value of 6e18 in HUVECs (FIG. 40). Internalization isreduced in HUASMCs for all sizes and surface charges (FIG. 41).PEGylated 100 nm nanospheres show the same level of internalization withHUVECs and HUASMCs with an approximate relative fluorescence value of 8.Negatively charged spheres for all sizes resulted in lessinternalization in both type of cells. Also, neutral, PEGylated andnegatively charged spheres of 1000 nm size had much less uptake for allthe time points. Overall, the interaction of 100 nm nanospheres withboth cell types result in a higher degree of internalization compared tothe 300, 500 and 1000 nm size nanospheres. Neutrally charged sphereseems to be more relevant than PEGylated and negatively charged spheresfor internalization. HUASMCs seem more resistant to internalization ofhollow spheres rather than HUVECs.

Blood Compatibility

A major challenge for the systemic delivery of synthetic vehicles forgene delivery is their lack of stability in the blood stream, theirdegradation, and clearance by the reticuloendothelial system, whichmakes the elucidation of their interaction with blood componentsessential. Several interactions with the family of spheres wereinvestigated to determine the potential systemic delivery in vivo of thehollow spheres. The nanoshells according to the invention were testedfor biocompatibility. The results are shown in FIGS. 22 to 25. Theeffect of various parameters such as the charge on the spheres and sizeof the spheres, on haemolysis and platelet activation was studied. Allthe experiments for haemocompatibility were performed using human venousblood from healthy volunteer donors.

The spheres according to the present invention are very suitable for invivo use due to their haemocompatibility. In fact, haemolysis graphs(FIGS. 22 and 23) demonstrate that chitosan/PGA spheres don't lysehaemoglobin (<1%). Moreover, this rate decreases for the smallest sizesand when the surface of the sphere is functionalised. Furthermore, theelisa detection experiment shows that the platelet activation decreasesafter functionalisation.

Haemolysis

Negatively charged spheres have a significantly higher % haemolysis atsizes 300, 500 and 1000 nm whereas PEGylated spheres have asignificantly reduced % haemolysis at these sizes. 100 nm spheres have asignificantly reduced % haemolysis for all surface charges (FIG. 42).Irrespective of size or surface charge, all spheres have a negligibleeffect on haemolysis (1%).

Platelet Activation

In this study, platelet activation was quantified by the release ofsoluble P-selectin (sP-Selectin) after incubation with all spheres. PBSwas used as a negative control. The results found that size does nothave a significant influence on the platelet activation. Negativelycharged spheres however, induce a significantly higher level ofsP-Selectin (p<0.05) when compared to other neutral and PEGylatedspheres for all sizes (FIG. 43).

Complement System

In this study complement activation was investigated by quantifying therelease of C3a after incubation with spheres. PBS was used as a negativecontrol and no significant difference was observed between samples andthe control (FIG. 44).

Plasma Clotting Time

Plasma recalcification profiles are used to mimic the intrinsiccoagulation system in vitro. PBS was used as a negative control in thisstudy. To quantify plasma recalcification profiles, T1/2 max wascalculated as the time at which half the saturate absorbance wasreached. Clotting times are significantly shorter (p<0.05) for allsamples when compared to the control (12.3+/−0.3 min). Negativelycharged spheres had a significantly decreased clotting time whencompared to other surface charges at 100 and 300 nm spheres. The absenceof a significant effect at larger sphere size indicates that there is asize at which surface charge does not have an effect. The effect of sizedid not have a significant effect on the clotting time of PEGylatedspheres whereas 300 nm spheres had a reduced clotting time when surfacecharge was negative and neutral when compared to 500 and 1000 nm spheres(FIG. 45).

The words “comprises/comprising” and the words “having/including” whenused herein with reference to the present invention are used to specifythe presence of stated features, integers, steps or components but doesnot preclude the presence or addition of one or more other features,integers, steps, components or groups thereof.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination.

The invention claimed is:
 1. A process for the preparation of a naturalor synthetic biodegradable polymeric hollow nanosphere comprising thesteps of: (i) providing a template comprising polymeric polystyrenebeads, mesoporous silica or diatomaceous silica; (ii) treating saidtemplate with a functionalising group to produce a functionalisedtemplate, (iii) treating said functionalised template with a solution ofone or more natural or synthetic biodegradable polymers selected fromthe group consisting of: collagen, elastin, chitosan, hyaluronan,alginate, polyesters, PEG-based polymers, dendritic or hyperbranchedpolymers, and combinations thereof, and agitating to form a polymericcoating on said template; (iv) cross-linking said coating on thetemplate with a cross-linking agent selected from the group comprising adendrimer, a hyper-branched dendritic polymer and linear polymericsystem, and combinations thereof, wherein the reactive groups in thecross-linking agent are —COOH or NH₂, groups and the reactive groups inthe polymer are —COOH or NH₂, the molar ratio of reactive —COOH or —NH₂groups in said dendrimer cross-linker to reactive groups —COOH or —NH₂in said polymer is in the range 50:1 to 1:50; and (v) removing thetemplate by treating said template with a solvent.
 2. A processaccording to claim 1, wherein the molar ratio of reactive —COOH or—NH₂groups in said dendrimer cross-linker to reactive groups —COOH or—NH₂, in said polymer is in the range 5:1 to 1:5.
 3. A process accordingto claim 2, wherein the polymer and crosslinkcr arc crosslinking using apromoter selected from the group comprising carbodiimide.transglutaminase, genepin, sulfonates, methyl sulfonate andtrifluoromethyl sulfonate, malernide, and EDC/NHS coupling.
 4. A processaccording to claim 1, wherein said functional ising group is selectedfrom the group consisting of sulphate, a carboxyl or amine group.
 5. Aprocess according to claim 4, wherein said fiinctionalising groupcomprises sulphate.
 6. A process according claim 1, wherein saidtemplate comprises sulfonated polystyrene beads.
 7. A process accordingto claim 1, wherein said dendrimer or said hyper-branched dendriticpolymer is selected from the group consisting of: peptide baseddendrimers, polyamidoamine (PAMAM), poly(2-dimethyl-aminoethylmethacrylate) (PDMAEMA), poly ethylene glycolmethyl ether methacrylate (PEGMEMA), ethylene dimethacrylate (EDGMA),polypropyleneimine, polyarylether, polyethyleneimine (PEI),poly-l-lysine (PLL), poly (ethylene glycol) methacrylate (PEGMA), poly(propylene glycol) methacrylate (PPGMA) polyacrylic acid and apolycarboxylic acid-polyethylene glycol-polycarboxylic acid (PPEGP)based dendrimer such as aconitic acid-polyethylene glycol-aconitic acidbased dendrimer (APEGA) and combinations thereof.
 8. A process accordingto claim 1, wherein the template is removed by treating thefunctionalised beads with acid solution in tetrahydrofuran (THF) priorto centrifugation to produce a nanosphere.
 9. A process according toclaim 1, further comprising encapsulating a biomolecule, therapeutic orimaging agent in said nanospheres.
 10. A process according to claim 9,wherein encapsulation is carried out by means of physical diffusion. 11.A process according to claim 10, wherein encapsulation is carried out bymeans of emulsification.
 12. A process according to claim 9, whereinsaid biomolecule, therapeutic or imaging agent is selected from thegroup consisting of pDNA, polyplexes, growth factors, peptides, viraland non-viral vectors (pDNA), doxorubicin, FITC, tryptophan, rhodamine,4′,6-diamidino-2-phenylindole (DAPI), TOPRO3, flourescein andderivatives thereof, red dyes, green dyes, AlexaFlor; a fluorescentprotein, as GFP/eGFP, YFP, and chemicals, APIs, drugs and pro-drugs. 13.A process according to claim 1, wherein the polymeric solution comprisesa polymer selected from the group consisting of collagen, elastin,chitosan, hyaluronan, alginate, PEG-based polymer and wherein thecrosslinker is a dendrimer selected from the group consisting of:peptide based dendrimers, polyamidoamine (PAMAM), poly (2-dimethyl-aminoethylmethacrylate) (PDMAEMA), polyethyleneglycolmethylethermethacrylate (PEGMEMA), ethylene dimethacrylate (EDGMA), poly-l-lysine(PLL), polyethyleneimine (PEI), polyglutamic acid (PGA), poly (ethyleneglycol) methacrylate (PEGMA) and poly (propylene glycol) methacrylate(PPGMA), (meth)acrylic acid monomers or NHS monomers withmulti-functional vinyl monomers, a polycarboxylic acid-polyethyleneglycol-polycarboxylic acid (PPEGP) based dendrimer such as aconiticacid-polyethylene glycol-aconitic acid based dendrimer (APEGA) andcombinations thereof.
 14. A process according to claim 1, furthercomprising the step of treating said polymer coated template with asecond polymeric solution comprising a polymer selected from the groupconsisting of polyglutamic acid, hyaluronic acid, alginate, PLGA,poly(caprolactone), and poly(d, l-lactide) to produce multilayerpolymeric nanospheres.